Paolo Macchiarini

Clinical transplantation of a tissue-engineered airway

BACKGROUND The loss of a normal airway is devastating. Attempts to replace large airways have met with serious problems. Prerequisites for a tissue-engineered replacement are a suitable matrix, cells, ideal mechanical properties, and the absence of antigenicity. We aimed to bioengineer tubular tracheal matrices, using a tissue-engineering protocol, and to assess the application of this technology in a patient with end-stage airway disease. METHODS We removed cells and MHC antigens from a human donor trachea, which was then readily colonised by epithelial cells and mesenchymal stem-cell-derived chondrocytes that had been cultured from cells taken from the recipient (a 30-year old woman with end-stage bronchomalacia). This graft was then used to replace the recipients left main bronchus. FINDINGS The graft immediately provided the recipient with a functional airway, improved her quality of life, and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. INTERPRETATION The results show that we can produce a cellular, tissue-engineered airway with mechanical properties that allow normal functioning, and which is free from the risks of rejection. The findings suggest that autologous cells combined with appropriate biomaterials might provide successful treatment for patients with serious clinical disorders.

Engineered whole organs and complex tissues

End-stage organ failure is a key challenge for the medical community because of the ageing population and the severe shortage of suitable donor organs available. Equally, injuries to or congenital absence of complex tissues such as the trachea, oesophagus, or skeletal muscle have few therapeutic options. A new approach to treatment involves the use of three-dimensional biological scaffolds made of allogeneic or xenogeneic extracellular matrix derived from non-autologous sources. These scaffolds can act as an inductive template for functional tissue and organ reconstruction after recellularisation with autologous stem cells or differentiated cells. Such an approach has been used successfully for the repair and reconstruction of several complex tissues such as trachea, oesophagus, and skeletal muscle in animal models and human beings, and, guided by appropriate scientific and ethical oversight, could serve as a platform for the engineering of whole organs and other tissues.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

SeriesEngineered whole organs and complex tissues

End-stage organ failure is a key challenge for the medical community because of the ageing population and the severe shortage of suitable donor organs available. Equally, injuries to or congenital absence of complex tissues such as the trachea, oesophagus, or skeletal muscle have few therapeutic options. A new approach to treatment involves the use of three-dimensional biological scaffolds made of allogeneic or xenogeneic extracellular matrix derived from non-autologous sources. These scaffolds can act as an inductive template for functional tissue and organ reconstruction after recellularisation with autologous stem cells or differentiated cells. Such an approach has been used successfully for the repair and reconstruction of several complex tissues such as trachea, oesophagus, and skeletal muscle in animal models and human beings, and, guided by appropriate scientific and ethical oversight, could serve as a platform for the engineering of whole organs and other tissues.

The first tissue-engineered airway transplantation: 5-year follow-up results

BACKGROUND In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results. METHODS The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen. FINDINGS By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life. INTERPRETATION These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.

Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

Primary tracheal tumours.

Primary tumours of the trachea can be benign or malignant and account for fewer than 0.1% of tumours. However, they are a diagnostic and therapeutic challenge. Benign tumours are usually misdiagnosed as asthma or chronic lung disease, and can delay diagnosis for months or years. Because of their rapid growth and onset of haemoptysis, malignant tumours are often diagnosed earlier than benign tumours and patients thus often present with locally advanced disease. Inappropriate treatment is an equally frustrating issue. Modern techniques for tracheal surgery-laryngotracheal, tracheal, or carinal resection-combined with radiotherapy, can be offered curatively with low perioperative risks. Nevertheless, the low numbers of patients undergoing resection and the associated poor survival in epidemiological studies over the past two decades have shown that surgery is rarely considered outside referral centres, with radiotherapy or another form of local treatment (eg, endotracheal stents, debridement, brachytherapy) generally preferred. The liberal use of these other techniques should be avoided because surgery has the potential to cure all patients with benign and low-grade tumours and most patients with malignant primary tracheal tumours, and other techniques are usually palliative at best.

Tissue engineered human tracheas for in vivo implantation

Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

Thymic neuroendocrine carcinoma (carcinoid): A clinicopathologic study of fourteen cases

The medical records and histologic documents of 14 patients treated at our institution for a thymic carcinoid tumor were reviewed. There were 3 women and 11 men with an age range from 35 to 71 years. One patient had a multiple endocrine neoplasia syndrome; another had a neurofibromatosis. Twelve tumors were revealed by local symptoms and two were asymptomatic. One patient had Cushings syndrome that appeared secondarily and was related to metastases. Tumors ranged from 6 to 20 cm and had the characteristic histologic appearance of atypical carcinoid tumor. Immunohistochemical evaluations were done. Tumors were positive for cytokeratin (92%), neuroendocrine markers (100%), and p53 oncoprotein (29%). S-100 protein antibody revealed numerous sustentacular cells in one case. Overall survival was 46% and 31% at 3 and 5 years, respectively. However, all patients died of the disease within 109 months as a result of local progression (n = 5), local relapse (n = 3), distant metastases (n = 8), or a combination of these reasons. Median survival was 71, 30, and 5 months for patients who had total resection (n = 4), partial resection (n = 5), or simple biopsy (n = 4), respectively (p = 0.023). In conclusion, thymic carcinoid tumors can be considered thymic neuroendocrine carcinomas because of their malignant behavior and histologic appearance of atypical carcinoid tumors. Complete surgical resection offers the best hope for long-term survival.

Experimental and clinical evaluation of a new synthetic, absorbable sealant to reduce air leaks in thoracic operations ☆ ☆☆

BACKGROUND Air leaks after pulmonary resections may contribute to increased patient morbidity, delayed removal of chest drainage tubes, and prolonged hospitalization. OBJECTIVE The purpose of this study was to investigate the effects of a new synthetic, absorbable sealant on the healing of healthy bronchial and lung tissues (experimental study) and its safety and efficacy to stop air leaks after lung resection (clinical study). METHODS Fifteen large white pigs underwent a left upper lobectomy. All parenchymal surgical sites were sealed; the bronchial stump was either stapled, sealed, or both (n = 5 each). In the clinical study, 26 consecutive patients were prospectively randomized, intraoperatively, to standard closure of parenchymal surgical sites with (n = 15) or without (n = 11) the sealant. RESULTS In the experimental study, no postoperative air leaks occurred, with intact bronchial closures and normal tissues at death. In the clinical study, 100% of intraoperative leaks were sealed versus 18% of control patients (P =.001). Although 77% (n = 10) of treated patients remained leak-free from the end of the operation to chest tube removal versus 9% (n = 1) of control patients (P =.001), there was no statistical difference in the duration of postoperative chest tube time, hospital stay, or cost. There were no acute or late undesirable side-effects related to the sealant application. CONCLUSIONS The surgical adhesive investigated here demonstrated a compelling safety profile and significant clinical efficacy to stop air leaks after lung resections.