Alessandro Gonfiotti

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.

Development of bioengineered human larynx.

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.

Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

Dynamic decellularization and cross-linking of rat tracheal matrix

Decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the biochemical and structural properties of the extracellular matrix are still to be found. For this aim, bioreactors could be an alternative means to dynamically treat the biological samples, automatically controlling all the variables involved in the process and speeding up the entire procedure in order to deal with a suitable scaffold within a limited time period. This paper presents the characterization of rat tracheae decellularized in dynamic conditions, implementing a detergent-enzymatic method, previously considered. Only 6 cycles were enough to generate a tracheal matrix that was histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers was well preserved, such as the epithelial cilia, the luminal basement membrane and the main matrix components. The elastin content decreased, even if not significantly, after the decellularization protocol. Mechanical properties of the treated tissues were slightly affected by the procedure, and were partially recovered after crosslinking with genipin, a naturally-derived agent. The use of bioreactors could enhance the decellularization procedure of tissues/organs, but a careful selection of the processing parameters is needed in order to prevent large modifications compared to the native condition.

Safety and Effectiveness of a New Fibrin Pleural Air Leak Sealant: A Multicenter, Controlled, Prospective, Parallel-Group, Randomized Clinical Trial

BACKGROUND This study evaluated the sealing capacity and safety of a new fibrin sealant (FS) to reduce alveolar air leaks (AALs) after pulmonary resections in a randomized controlled clinical trial conducted in 3 Italian centers. METHODS The study randomized (1:1) 185 patients with an intraoperative AAL graded 1 to 3 according to the Macchiarini scale: 91 received FS and 94 had standard lung closure. The primary outcomes were the length of postoperative AAL duration and the mean time to chest drain removal. Other end points included the percentage of patients without AAL, the development of serum antibodies against bovine aprotinin, and any adverse event related to FS. Chest drains were removed when fluid output was 100 mL/day or less, with no air leak. RESULTS The study groups were comparable with respect to demographic variables and surgical procedures. The FS group showed a statistically significant reduction in duration of postoperative AALs (9.52 vs 35.8 hours; p < 0.005) and in the percentage of patients with AALs at wound closure (81.11% vs 100%; p < 0.001); the difference in time to chest drain removal was not significant. Pleural empyema developed in 1 patient with FS treatment vs in 4 with standard treatment, and antibodies against bovine aprotinin were found in 34 of 91 FS-treated patients. CONCLUSIONS The present study showed that the new FS is safe and effective in preventing AALs after lung resections and in shortening the duration of postoperative AALs.

Adverse effects of fibrin sealants in thoracic surgery: the safety of a new fibrin sealant: multicentre, randomized, controlled, clinical trial

OBJECTIVES The safety of fibrin sealants (FS) has been questioned in the light of recent reports of adverse effects. We evaluated the safety of a new FS in a randomized controlled trial (RCT). METHODS Multicentre, open-label Phase II/III RCT to evaluate the safety of the new FS. The trial was approved by the Ethic Committee of each three participating Centre. FS includes two components (component 1: fibrinogen; component 2: thrombin), each of them subjected to two viral inactivation procedures. Out of 200 screened patients, 185 eligible patients (49 females, 136 males), aged between 18 and 75 years, undergoing major thoracic surgery were randomized to receive FS (#91 patients) as an adjuvant for air leak control or no treatment (#94 patients, control group). Safety variables were: percentage of subjects with adverse events associated with the therapy; formation of antibodies against bovine aprotinin; vital signs (blood pressure, body temperature, heart and respiratory rate); laboratory parameters. RESULTS Overall operative mortality was 3.2% (6/185), 1.1% in the FS group and 5.3% in the control group, respectively. Twenty patients (22%) had adverse events in the FS group and 22 (23.4%) in the control group. Atrial fibrillation (five patients in the FS group and four in the control group) and hyperpyrexia (five and seven patients, respectively, in the two groups) were the most common adverse events. No patient reported thromboembolic events (pulmonary embolism or deep vein thrombosis) during the in hospital stay or within 1 month from discharge. None of the adverse events was considered as treatment related. The formation of bovine aprotinin antibodies was reported in a total of 34 patients (37.4%) in the FS group and was not related to any adverse effect. CONCLUSIONS The present RCT did not show any increased risk of adverse events, and of surgical complications, related to the use of the new FS.

RETRACTED: Development and Validation of a New Outcome Score in Subglottic Stenosis

BACKGROUND We prospectively evaluated a clinical and endoscopic score, the tracheal endoscopic clinical score (TECS), developed as a disease-specified outcome measure in adult patients undergoing operation for subglottic stenosis. We also performed a retrospective chart review to identify preoperative and intraoperative risk factors for worse TECS. METHODS The TECS includes endoscopic (vocal cord and glottic function, anastomotic healing, and patency) and interview (respiration, voice, swallow) variables, and was administered at 6-month follow-up. Endoscopic and subjective domains were weighted to obtain a continuous TECS index ranging from 0 (best) to 1 (worse). The TECS and preoperative variables relationships were evaluated by univariate and multivariate analysis. RESULTS We collected data (January 2009 to December 2010) from 30 patients (mean age, 48.3±19 years) undergoing subglottic resection and primary reconstruction. Stenosis etiology was postintubation (n=8), idiopathic (n=2), tracheostomy (n=18), and malignant (n=2). Surgery included Pearson operation with (n=7) or without (n=23) a Liberman-Mathisen cricoplasty. Mean length of resected trachea was 30.5±13.5 mm, and mean hospital stay was 7.4 days. Mortality rate was 1 patient (3.3%). The univariate analysis showed positive correlation between 6-month TECS and degree of stenosis (McCaffrey and Cotton scale 0 to 4) stage 4, tracheostomy or T-tube at surgery, bottleneck-type transition stenosis, and resection length. At multivariate analysis, the presence of tracheostomy, bottleneck-type transition stenosis and resection length were indicators of worse postoperative functional result. CONCLUSIONS The TECS seems to be a valid and simple instrument to identify preoperative variables predicting worse results and to assess postoperative outcome. Validation on larger series is necessary.

Robot-sewn Ivor-Lewis anastomosis: preliminary experience and technical details

Robot‐assisted minimally invasive esophagectomy with intra‐thoracic anastomosis showed encouraging results but there is a lack of data to demonstrate the safety and feasibility.

Uniportal thoracoscopic decortication for pleural empyema and the role of ultrasonographic preoperative staging

Objectives The surgical approach to chronic pleural empyema is still controversial. Video-assisted thoracic surgery (VATS) debridement and decortication has shown favourable outcomes, while the uniportal VATS (U-VATS) approach is still anecdotal. We report our experience with ultrasonographic (US) preoperative staging followed by U-VATS decortication for pleural empyema. Methods We performed a retrospective analysis of patients who underwent surgical treatment of stage II and stage III pleural empyema from 2012 to 2015. Pre-, intra- and postoperative data were investigated to evaluate outcomes including postoperative complications and disease recurrence. Results were analysed according to preoperative US appearance of pleural space (stages A-E) and surgical approach (thoracotomy vs U-VATS). Results We performed 30 (47%) uniportal thoracoscopic pleural decortication and 34 (53%) open decortication for empyema in stage II (40%) or III (60%) obtaining a complete debridement and decortication in all patients. In-hospital mortality was zero and overall morbidity was 29%. U-VATS was associated with lower blood loss (118 ±  80 ml vs 247  ±  140 ml P  < 0.001), lower chest tubes duration (5.6 ±  1.4 vs 10.6  ±  4.4 days P  < 0.001), shorter hospital stay (6.7  ±  1.9 vs 12.2  ± 4.7 days, P  < 0.001) and lower complications (10% vs 16%, P  < 0.001). Elevated US patterns (D-E) are associated with thoracotomy, higher blood loss, operative time and a significant incidence of complications. Conclusions Uniportal thoracoscopic decortication for pleural empyema is a safe and effective approach for selected patients based on a combination of clinical and imaging staging. US patterns well corresponded with intraoperative pleural findings and showed a prognostic value.

Hybrid treatment of T3 chest wall lung cancer lobectomy

Nowadays the treatment of patients with non-small cell lung cancer (NSCLC) that invades the chest wall is still questioned. The classic approach is a lobectomy that requires chest wall resection through thoracotomy, but thanks to the progress in the field of thoracoscopic surgery, this procedure can be performed by video-assisted thoracoscopic surgery (VATS). Major advances have been made in recent years both in the surgical technique associated with thoracoscopy and in the instrumentation available today. This has allowed the use of thoracoscopic technique even in advanced disease. To choose to perform complex surgery in assisted video surgery, considerable experience is needed to avoid making mistakes and giving up a better approach for the patient only for any technical difficulties. Thoracoscopy is not currently the preferred intervention for patients with chest wall invasion because there are insufficient studies on the feasibility of lobectomy with thoracoscopic wall resection, although the thoracoscopic approach has reduced mortality and morbidity in lung cancer cases not in advanced stage. We discuss our experience in three patients using hybrid approach with assisted video thoracoscopic lobectomy and a chest wall en-block resection with an alternative method of estimating thoracic wall resection that uses assisted video surgery and hypodermic needles (minimally invasive posterior approach).