Different bioengineering techniques have been applied repeatedly for the reconstruction of extensive airway defects in the last few years. While short-term surgical success is evident, there is a lack of long-term results in patients. Here, we report the case of a young male who received a 5x2 cm bioartificial airway patch for tracheoesophageal reconstruction focusing on clinical defect healing and histomorphological tissue reorganization 2.5 years after surgery. We generated bioartificial airway tissue using a cell-free biological vascularized scaffold that was re-endothelialized and reseeded with the recipient's autologous primary cells and we implanted it into the recipient's left main bronchus. To investigate host-integration 2.5 years after the implantation, we obtained biopsies of the implant and adjacent tracheal tissue and processed these for histological and immunohistochemical analyses. The early postoperative course was uneventful and the transplanted airway tissue was integrated into the host. 2.5 years after transplantation, a bronchoscopy confirmed the scar-free reconstruction of the former airway defect. Histological work-up documented respiratory airway mucosa lining the bronchial reconstruction, making it indistinguishable from native airway mucosa. After transplantation, our bioartificial airway tissue provided perfect airway healing, with no histological evidence of tissue dedifferentiation.

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver.

Objective: In the treatment of advanced stages of lung cancer, increasingly more multimodality approaches applying radiotherapy and/or chemotherapy in a neo-adjuvant setting are being introduced. The impact of induction therapy, especially radiotherapy, on bronchial tissue viability has not been investigated so far. Methods: In 2008, we determined the tissue viability of bronchial segments obtained during surgery in 45 consecutive patients, including patients after neo-adjuvant radiochemotherapy (RCTX). Bronchial tissue viability was analysed by histology, life-dead assay and cell proliferation in tissue-specific culture media. Biomedical findings were compared with the clinical course of the patients. Results: Tissue samples of 44 patients were included into this study. Fourteen patients (32%) had undergone neo-adjuvant RCTX. Histology and life-dead assay of the bronchial segments did not show significant differences. While patient age, sex, tumour entity and site of resection had no influence on cell proliferation in vitro, previous RCTX resulted in a 46% decrease of bronchial tissue viability (P = 0.01). However, this effect was not reflected by the clinical course of the operated patients. Conclusions: Neo-adjuvant RCTX reduces bronchial tissue viability substantially. However, this impairment does not necessarily translate into an increased rate of postoperative bronchial insufficiencies. Standard histological work-up is not sensitive enough to characterise changes in bronchial tissue viability following RCTX. (C) 2009 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V All rights reserved.

BACKGROUND:The lack of transplant vascularization forecloses the generation and clinical implementation of bioartificial tissues. We developed techniques to generate a bioartificial human tissue with an innate vascularization. The tissue was implanted clinically as proof of concept to evaluate vascular network thrombogenicity and tissue viability after transplantation.METHODS:A porcine small bowl segment was decellularized in a two-step procedure, preserving its vascular structures. The extracellular matrix was characterized quantitatively for DNA residues and protein composition. The vascular remainings were reseeded with human endothelial cells in a dynamic tissue culture. The engineered tissue was characterized by (1) histology, (2) immune-histology, (3) life-dead assay, and (4) metabolic activity. To evaluate the tissue capabilities, it was implanted clinically and recovered after 1 week.RESULTS:Tissue preparation with sodium desoxycholate monohydrate solution resulted in an incomplete decellularization. Cell residues were removed by additional tissue incubation with DNAse. The human endothelial cells formed a viable endothelium inside the primarily porcine extracellular matrix, expressing CD31, Flk-1, and vascular endothelium-cadherin. The metabolic activity of the bioartificial tissue increased continuously over time in vitro. Clinical tissue transplantation confirmed vessel patency and tissue viability for 1 week.CONCLUSIONS:The feasibility to bioengineer a human tissue with an innate vascularization has been shown in vitro and the clinical setting. These results may open the door for the clinical application of various sophisticated bioartificial tissue substitutes and organ replacements.

Introduction: Three dimensional cell systems have been already established for the cornea and the skin. These models are – due to their organ-specific properties – suitable for the analysis of different substances with regard to their biocompatibility and toxicology which up to now could be examined only by animal experiments. The airway represents also an important entry gate for many different pathogenic agents and environmental impacts. A three dimensional model of the trachea becomes also more and more important as an alternative for animal experiments because it shows similarities in structure and function of the human trachea. Aim: Our aim is to develop a functional trachea test system in a bioreactor system in which the cells are cultivated under physiological conditions and simulation of the respiration. This model could be used as a test system for the analysis of different substances (e.g. nanoparticles or other aersols) as an alternative for preclinical animal testing and it could be also used as a biological graft. Materials and Methods: For the assembly of a 3D-model of the trachea, we use human respiratory epithelium cells. The isolation occurs with the sprouting method from small tissue pieces of a biopsy. According to confluence the cells were trypsinated and used for the construction of a three dimensional model. For further colonization the cells were seeded in Collagen-I coated inserts and cultivated in an air-liquid interface on different membranes to mimic the native conditions. The cultivation occurs with special Airway Epithelial Cell Growth Medium. The cells were characterized via histological and immuno-histological methods. Results: The picture below shows a piece of the biopsy with grown-out respiratory cells. In preliminary tests human respiratory cells were cultivated on inserts with a PETand a biological membrane. Most of these cells are vital and show a high rate of proliferation. There are ongoing experiments with a co-culture of fibroblast cells and respiratory epithelium cells. The cells were also cultivated in a special trachea-reactor with simulation of the respiration to stimulate the ciliary activity to adjust the mucociliary clearance which is one of the major tasks of the trachea. Perspectives: The cultivation of functional epithelium cells with active cinocilia and mucus secretion is very important for the development of a functional test system. Therefore a better understanding of cells and matrix and also the stimuli for the differentiation of these cells is immanently necessary. The next step is the cultivation of the respiratory cells in co-culture with fibroblasts in a bioreactor system under mechanical stimulation and simulation of the respiration.