EPR spectroscopy solutions for assessment of decellularization of intrathoracic organs and tissues.

Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.

Laryngeal transplantation in minipigs: early immunological outcomes.

Despite recent tissue-engineering advances, there is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. A recent clinical transplant was a success. Using quantitative immunofluorescence targeted at immunologically relevant molecules, we have studied the early (48 h and 1 week) immunological responses within larynxes transplantated between seven pairs of National Institutes of Health (NIH) minipigs fully homozygous at the major histocompatibility complex (MHC) locus. There were only small changes in expression of some molecules (relative to interindividual variation) and these were clearest in samples from the subglottic region, where the areas of co-expression of CD25(+) CD45RC(-) CD8(-) and of CD163(+) CD172(+) MHC-II(-) increased at 1 week after transplant. In one case, infiltration by recipient T cells was analysed by T cell receptor (TCR) Vß spectratype analysis; this suggested that changes in the T cell repertoire occur in the donor subglottis mucosal tissues from day 0 to day 7, but that the donor and recipient mucosal Vß repertoires remain distinct. The observed lack of strong immunological responses to the trauma of surgery and ischaemia provides encouraging evidence to support clinical trials of laryngeal transplantation, and a basis on which to interpret future studies involving mismatches.

Tissue-engineered airway: a regenerative solution.

The use of synthetic degradable or permanent polymers and biomaterials has not yet helped to achieve successful clinical whole-airway replacement. A novel, clinically successful approach involves tissue engineering (TE) replacement using three-dimensional biologic scaffolds composed of allogeneic extracellular scaffolds derived from nonautologous sources and recellularized with autologous stem cells or differentiated cells. In this paper, we discuss this novel approach and review information that can lead to a better understanding of stem cell recruitment and/or mobilization and site-specific tissue protection, which can be pharmacologically boosted in humans.

Laryngeal transplantation in minipigs: vascular, myologic and functional outcomes.

There is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. Regenerative medicine offers promise, but cannot presently deliver implants with functioning neuromuscular units. A single well-documented laryngeal transplant in man was a qualified success, but more information is required before clinical trials may be proposed. We studied the early response of the larynx to laryngeal transplantation between 17 pairs of NIH minipigs full matched at the MHC2 locus. Following iterative technical improvements, pigs had good swallowing and a patent airway at 1 week. No significant changes in mucosal blood flux were observed compared with pre-operative measurements. Changes in muscle morphology and fibre phenotype were observed in transplant muscles retrieved after 7 days: the levels of fast and slow myosin heavy chain (MyHC) protein were reduced and embryonic MyHC was up regulated consistent with denervation induced atrophy. At 1 week laryngeal transplantation can result in good swallowing, and is not associated with clinical evidence of ischemia-reperfusion injury in MHC-matched pigs.

Artificial lung: current perspectives.

While the number of the patients suffering from end-stage pulmonary disease has been increasing, the most common treatment for this entity remains mechanical ventilation that entails the risks of lung damage by itself. Although the lung protective strategy for the prevention of further damage to the lung tissue has been elucidated and performed, mechanical ventilation alone as the management tactic coping with the patients of acute respiratory distress syndrome, chronic respiratory failure and lung transplantations has been a frustrated scenario. Extracorporeal membrane oxygenation or extracorporeal lung assist have been applied to these patients with occasional success, but it always accompanies difficulties such as multiple blood transfusion, labor intensity, technically complexity and tendency to infection. In contrast to advances in the development of cardiac or renal support systems for adults, the development of extra-, para- and intracorporeal mechanical systems for acute or chronic lung respiratory failure has logged far behind. It has been mostly due to the lack of the capable technologies. Entering 21st century with advent of new technology especially invention of the low resistance oxygenator, the developments of artificial lungs have entered the new stage. In this report current status of the artificial lungs will be reviewed.

Early immunological changes associated with laryngeal transplantation in a major histocompatibility complex-matched pig model.

Laryngeal transplantation is an increasingly viable proposition for patients with irreversible diseases of the larynx. One human transplant has been performed successfully, but many questions remain before routine transplantation can begin. In order to measure the immunological changes in mismatched transplants, it is first necessary to know the immediate combined effects of ischaemia-reperfusion injury (IRI) plus the added insult of major surgery in a fully matched setting. We measured the changes in immunologically active mucosal cells following 3 h of cold ischaemia and 8 h of in situ reperfusion in a major histocompatibility complex (MHC)-matched minipig model (n = 4). Biopsies were prepared for quantitative, multiple-colour immunofluorescence histology. The number of immunologically active cells was significantly altered above (supraglottis) and below (subglottis) the vocal cords following transplantation and reperfusion (P < 0.05, P < 0.001, respectively). However, the direction of the change differed between the two subsites: cell numbers decreased post-transplant in the supraglottis and increased in the subglottis. Despite the statistical evidence for IRI, these changes were less than the large normal inter- and intrapig variation in cell counts. Therefore, the significance of IRI in exacerbating loss of function or rejection of a laryngeal allograft is open to question. Longer-term studies are required.

Generation of a bioartificial fibromuscular tissue with autoregenerative capacities for surgical reconstruction.

INTRODUCTION: Anecdotal clinical reports denote first tissue engineering applications entering medical practice. Currently it is still unknown, if these new types of implants will tolerate the specific needs in cancer patients undergoing postoperative chemo- and radiotherapy. METHODS: We implemented a radiotherapy protocol (cumulative dosis 40 Gy) on generated human bioartificial fibromuscular tissues in vitro. We monitored tissue vitality during radiotherapy and tissue recovery (8 weeks follow up period) applying histological methods. RESULTS: The biopsy procedure and seeding techniques yielded a viable 3 dimensional bioartificial human tissue. Radiation resulted in immediate devitalization without destroying tissue integrity. The bioartificial tissue recovered entirely in vitro within 6 weeks. CONCLUSION: Bioartificial human implants appear applicable for surgical reconstruction in oncologic patients potentially facing postoperative radiotherapy.

Model for experimental revascularized laryngeal allotransplantation.

BACKGROUND: Although a human laryngeal transplant has been undertaken successfully, important questions remain that require a suitable animal model. METHODS: A pig model for allotransplantation has been developed. Organ perfusion was studied in nine animals before four transplants were performed in congenic (unrecovered) animals and eight in unmatched (recovered) animals. Larynges were regularly examined endoscopically until death at 14 days. Immunosuppression included the use of tacrolimus. Revascularization was achieved by anastomosing the donor right cervical vascular tree to the recipient common carotid. In recovered animals, four allografts were placed orthotopically and four heterotopically. RESULTS: The pig larynx was perfused adequately via the right cervical vascular tree and congenic grafts were well tolerated. Of eight allografts, seven were well tolerated and remained healthy for the duration of the study (14 days). One allograft became infected between days 4 and 7 after operation. Median operating time was 6 h, with a median cold ischaemia time of 3 h. CONCLUSION: Revascularized allotransplants of the larynx can be undertaken reliably in pigs and this provides a preclinical model for studies of laryngeal transplantation.

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models.

Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig-to-human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate-anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de-oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (deltapO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK 15 assay DXS 5000 led to a complete, dose-dependent inhibition of human serum cytotoxicity with an average IC50 of 43 +/- 18 microg/ml (n=8). Pig lungs perfused with untreated human blood (n=2) underwent HAR within 105 +/- 64 min, characterized by increased PVR, decrease of deltapO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n=4) prolonged lung survival to 170 +/- 14 min for 1 mg/ml and 250 +/- 42 min for 2 mg/ml. and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n=2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti-Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig-to-human xenotransplantation models and therefore a candidate for complement-inhibitory/anti-inflammatory therapy either alone or in combination with other substances and warrants further investigation.

Partial cricoidectomy with primary thyrotracheal anastomosis for postintubation subglottic stenosis.

OBJECTIVE: We describe a Pearson-type technique and evaluate its results for postintubation subglottic stenosis. METHODS: Forty-five patients underwent a partial cricoidectomy with primary thyrotracheal anastomosis, and 5 underwent simultaneous repair of a tracheoesophageal fistula as well. Twenty-four (53%) patients were referred to us after initial conservative (n = 21) or operative (n = 3) management. There were 27 cuff lesions, 7 stomal lesions, and 11 at both levels. The upper limit of the stenosis was 1.5 cm (range, 1-2.5 cm) below the cords, and the subglottic diameter was reduced by 60% in 38 (84%) of the patients. The length of airway resection ranged from 2 to 6 cm (median, 3 cm). Despite 23 thyrohyoid or suprahyoid releases, 8 anastomoses were under tension. RESULTS: Thirty-seven (82%) patients were extubated after the operation (n = 30) or within 24 hours (n = 7). Six patients required postoperative airway stenting (median, 5.5 days). Early (<30 days) complications occurred in 18 (41%) patients, mainly as transient airway and voice complaints, aspiration, and dysphagia. One (2%) patient died of myocardial infarction. Late morbidities were 2 failures occurring as bilateral recurrent nerve paralysis and restenosis requiring definitive tracheostomy. Patients had excellent or good anatomic (n = 42 [96%]), functional (n = 41 [93%]), or both types of long-lasting results, with no stenotic relapse. CONCLUSIONS: Partial cricoidectomy with primary thyrotracheal anastomosis can be applied in patients with postintubation stenosis extending up to 1 cm below the cords and measuring up to 6 cm in length with excellent-to-good definitive results. The association with a tracheoesophageal fistula does not contraindicate surgical repair.

Transcervical-transtracheal endoluminal repair of membranous tracheal disruptions.

Tracheal lacerations are rare and potentially hazardous complications of tracheal intubation. Surgical repair is the treatment of choice of tracheal injuries although nonoperative management is occasionally appropriate for well-selected patients. We describe our personal technique of anterior transcervical-transtracheal endoluminal suture of iatrogenic lacerations of the membranous trachea and our results with this approach in 8 patients. This method is less invasive than conventional cervical or transthoracic approaches.

Factors affecting long-term survival after en-bloc resection of lung cancer invading the chest wall.

OBJECTIVE: Several reports emphasize the importance of en-bloc resection as the optimal surgical treatment of lung cancer with chest wall invasion. We investigated possible factors which could affect long-term survival following radical resection of these tumors. METHODS: Between 1981 and 1998, 100 patients (90 male; ten female), with a median age of 60 years (36-84), underwent radical en-bloc resection of non-small cell lung cancer (NSCLC) with chest wall involvement. Patients with superior sulcus tumors invading the thoracic inlet were excluded from this series. There were 43 squamous and 57 non-squamous tumors. The median number of resected ribs was three (1-5). Lung resection included 73 lobectomies, two bilobectomies, 18 pneumonectomies and seven segmentectomies. Chest wall resection also extended to the sternum in one patient, the transverse process in one, the costotransverse foramen and hemivertebrae in two. All patients had a complete resection. Sixty-three patients received postoperative radiotherapy and 12 received chemotherapy. Histological data, including differentiation and depth of chest wall invasion, were carefully reviewed. The effect of various factors on survival were studied. RESULTS: There were four in-hospital deaths. Lymph node involvement was negative on surgical specimens in 65 patients, and 28 patients had positive N1 nodes; the final histology revealed seven N2 diseases. Chest wall invasion was limited to the parietal pleura in 29 patients and included intercostal muscles, bones and extrathoracic muscles in 67, 24 and seven cases, respectively. The overall 2-year survival rate was 41%. The 5-year survival for patients with N0, N1 and N2 disease was 22, 9 and 0%, respectively. A local recurrence occurred in 13 patients, with four having a new resection and 45 patients developing systemic metastases. The nodal status (N0-1 vs. N2; P=0. 026) and the number of resected ribs(<2 vs. >2; P=0.03) were survival predictors in univariate analysis. By multivariate analysis, the two independent factors affecting long-term survival were the histological differentiation (well vs. poorly differentiated; P=0. 01) and the depth of chest wall invasion (parietal pleura vs. others; P=0.024). CONCLUSIONS: Histological differentiation and depth of chest wall involvement were the main factors affecting long-term survival in this series. The role of induction chemotherapy for tumors with poor prognosis should be investigated.

Surgical treatment of pulmonary aspergilloma: current outcome.

OBJECTIVE: This retrospective study was designed to confirm that aggressive pulmonary resection can provide effective long-term palliation of disease for patients with pulmonary aspergilloma. METHODS AND RESULTS: From 1959 to 1998, 84 patients underwent a total of 90 operations for treatment of pulmonary aspergilloma in the Marie-Lannelongue Hospital. The mean follow-up period was 9 years, and 83% of the patients were followed up for 5 years or until death, if the latter occurred earlier. The median age was 44 years. The most common indications were hemoptysis (66%) and sputum production (15%). Fifteen patients (18%) had no symptoms. Tuberculosis and lung abscess were the most common underlying causes of lung disease (65%). The procedures were 70 lobar or segmental resections, 8 cavernostomies, and 7 pneumonectomies. Five thoracoplasties were required after lobectomy (3 patients) or pneumonectomy (2 patients). The operative mortality rate was 4%. The major complications were bleeding (23 patients), prolonged air leak (31 patients), respiratory failure (10 patients), and empyema (5 patients). The actuarial survival curve showed 84% survival at 5 years and 74% survival at 10 years. During the first 2 years, death was related to the surgical procedure and the underlying disease. In contrast, 85% of the survivors had a good late result. CONCLUSION: Lobar resection in both the symptomatic and the asymptomatic patients was conducted in low-risk settings. For patients whose condition is unfit for pulmonary resection, cavernostomy may need to be undertaken despite the high operative risk. The better survival rate in this study may have been due to the selection of patients with better lung function and localized pulmonary disease.

Evaluation and outcome of different surgical techniques for postintubation tracheoesophageal fistulas.

OBJECTIVE: We evaluated the outcome of different surgical techniques for postintubation tracheoesophageal fistula. METHODS: Thirty-two consecutive patients aged 51 +/- 23 years had tracheoesophageal fistulas resulting from a median of 30 days of mechanical ventilation via endotracheal (n = 12) or tracheostomy (n = 20) tubes. Tracheoesophageal fistulas were 2.5 +/- 1.2 cm long and were associated with a tracheal (n = 10) or subglottic (n = 3) stenosis in 13 patients. RESULTS: All but 3 patients were weaned from respirators before repair. All operations were done through cervical incisions and included direct division and closure (n = 9), esophageal diversion (n = 3), muscle interposition (n = 6), or, more recently, tracheal or laryngotracheal resection and anastomosis with primary esophageal closure (n = 14). Nine thyrohyoid and two supralaryngeal releases reduced anastomotic tension. Twenty-three patients (74%) were extubated after the operation (n = 16) or within 24 hours (n = 7), and 7 required a temporary tracheotomy tube. One postoperative death (3%) was associated with recurrent tracheoesophageal fistula. Seven complications (22%) included recurrent tracheoesophageal fistula (n = 1), delayed tracheal stenosis (n = 2), dysphagia (n = 2), and recurrent nerve palsy (n = 2). Complications necessitated reoperation (n = 1), dilation (n = 2), definitive tracheostomy (n = 1), Montgomery T tubes (n = 1), and Teflon injection of the vocal cords (n = 1). Twenty-nine patients (93%) had excellent (n = 24) or good (n = 5) anatomic and functional long-term results. Complications have been less common (7% vs 38%) and long-term results better (93% vs 65%) recently with tracheal or laryngotracheal resection and anastomosis with primary esophageal closure as compared with previous procedures. CONCLUSIONS: Postintubation tracheoesophageal fistula is usually best treated with tracheal or laryngotracheal resection and anastomosis with primary esophageal closure even in the absence of tracheal damage.

Use of embryonic human trachea grown in nude mice to patch-repair congenital tracheal stenosis.

BACKGROUND: Long congenital tracheal stenosis is a life-threatening condition, and the available surgical treatments do not give satisfactory long-term results. METHODS: Human embryonic tracheas were implanted in the abdominal cavities of nude mice until their differentiation was completed. These differentiated tracheas were used to patch-repair surgically induced tracheal stenosis in piglets. The human, mouse, or pig origin, of all the cells in the two successive xenotransplants in the nude mouse and the pig, was determined on tissue sections by in situ hybridization with species-specific DNA probes. RESULTS: The transplanted pigs thrived and reached normal adulthood, irrespective of the administration of immunosuppressive treatment. The human tracheal tissue developed in nude mice conserved human structures, with the exception of feeding capillaries, which were of mouse origin. The tracheal patch in the adult healthy pigs comprised only pig cells organized into a fibrous scar, which was covered by normal pig epithelium. CONCLUSIONS: Results suggest that human embryonic trachea grown in nude mice can be successfully used as patch tracheoplasty for long congenital tracheal stenosis without conventional immunosuppression.

A preclinical model for laryngeal transplantation: anatomy and mucosal immunology of the porcine larynx.

BACKGROUND: A major step in translating work on laryngeal transplantation into clinical practice is the establishment of a preclinical model. We have investigated the anatomy and mucosal immunology of the porcine larynx in eight Minnesota Minipigs (12-37 weeks). METHODS: Neck dissections were carried out and the vascular tree was mapped. Snap-frozen biopsies from epiglottis, supraglottis, glottis, and subglottis were prepared for conventional histology, immunohistochemistry (CD45), and single and two-color immunofluorescence (CD3, MHC-II, CD45). RESULTS: The anatomy of the laryngeal skeleton was broadly similar to that of the human larynx. The blood supply is predominantly via the caudal thyroid vessels, with assistance from the cranial laryngeal artery. The porcine larynx is clearly highly immunologically active. Structured collections of leukocytes were found in the mucosal epithelium, around tubuloacinar glands, and occasionally in the submucosa. MHC-II and CD 3 cells were predominantly found within the epithelium. The highest densities of all cell types were observed in the epiglottis, tailing off caudally. The lowest densities were seen in the vocal cords. CONCLUSIONS: The porcine larynx is both anatomically and immunologically similar to the human larynx and contains a high level of immunological organization. It presents an ideal preclinical model for laryngeal transplantation.