Biomedical subjects
Thorsten Walles
Publications and source records attributed to Thorsten Walles.
Technical innovations of carinal resection for nonsmall-cell lung cancer.
BACKGROUND: We present our perioperative management of operable nonsmall-cell lung cancer invading the tracheobronchial bifurcation and the results obtained. METHODS: Fifty consecutive patients undergoing carinal surgery with radical lymphadenectomy over a 5-year period were studied. RESULTS: Eighteen patients (36%) were N2 and had chemoradiation (48 +/- 6 Gy) preoperatively. Surgery included 34 carinal pneumonectomies (24 right, 10 left), 11 carinal lobectomies (n = 6) or bilobectomies (n = 5), and 5 carinal resections, with (n = 3) and without (n = 2) reconstructions. Patients were ventilated through low tidal volume controlled techniques except during airway resection and reconstruction, during which the apneic (hyper) oxygenation techniques were used. High inspiratory oxygen concentrations, multiple collapse and reexpansions, hypoperfusion of the ipsilateral lung, and fluid overload were avoided. All patients but 1 were extubated in the operating room, 7 +/- 5 minutes after skin closure. Operative mortality (less than 30 days) and morbidity were 4% (n = 2) and 37% (n = 18), respectively. All resections but 1 (98%) R1 were complete. The number of resected nodes per patient was 9 +/- 2, and 7 (22%) of the 32 patients who had negative preoperative positron emission tomography results had micrometastatic mediastinal nodes. With a median follow-up of 38 months, actuarial 5-year and disease-free survivals were 51% and 47%, respectively. Disease-free survival was significantly affected by endobronchial extension (tracheobronchial angle invasion versus less than 0.5 cm from carina, p = 0.03) and nodal status (N0 versus N1-2, p = 0.02) in the multivariate analysis. CONCLUSIONS: Preoperative chemoradiation, carinal lobectomy, or left pneumonectomy, and radical lymphadenectomy do not worsen the therapeutic index of carinal surgery. The high incidence of micrometastatic nodes in positron emission tomography-negative patients justifies routine mediastinoscopy and radical lymphadenectomy.
Tissue remodeling in a bioartificial fibromuscular patch following transplantation in a human.
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A multifunctional bioreactor for three-dimensional cell (co)-culture.
Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown. Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4',6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14. The optimized seeding density in the matrix was 2.0 x 10(7) cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2-183.7 +/- 18.4 mmHg) and physiological pH values (7.4 +/- 0.02) and a constant level of pCO2 (43.1 +/- 2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82 +/- 6.7% living cells. No significant differences were found between spatial separated bioreactor compartments. Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.
Engineering of a vascularized scaffold for artificial tissue and organ generation.
Tissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However, complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized by quantitative 2-[18F]-fluoro-2'-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistological work up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascular structures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding. Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate that our generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinical application.
Biological vascularized matrix for bladder tissue engineering: matrix preparation, reseeding technique and short-term implantation in a porcine model.
PURPOSE: We generated a vascularized, autologous, reseeded bladder substitute and evaluated immediate vascularization and perfusion of the graft after implantation to the recipient organism in a porcine model. MATERIAL AND METHODS: Acellular matrix was processed from porcine small bowel segments by subsequent mechanical, chemical and enzymatic decellularization, preserving the jejunal arteriovenous pedicles. In 2 separate steps the matrix was reseeded with primary bladder smooth muscle cells (SMCs) and urothelial cells (UCs), and its vascular structures were resurfaced with endothelial progenitor cells (EPCs). To evaluate graft perfusion short-term implantation was performed. RESULTS: The acellular scaffold was successfully repopulated with multilayers of ingrowing SMCs and superficial UCs. After reseeding the jejunal arteriovenous pedicles with EPCs and cultivation for 3 weeks the larger vessels as well as the intramural scaffold capillary network were repopulated with cell monolayers expressing endothelial specific proteins. Perfusion stagnation and implant thrombosis occurred within 30 minutes after the implantation of acellular scaffolds not reseeded with EPCs. In the EPC reseeded group the vascular system revealed intact perfusion and no relevant thrombus formation was observed after 1 or 3 hours. CONCLUSIONS: The current study of successful SMC and UC reseeding, vessel resurfacing with EPCs and short-term vascular patency represents the promising in vitro and in vivo basis for further evaluation of this biological vascularized matrix in chronic long-term large animal implantation experiments.
Functional neointima characterization of vascular prostheses in human.
BACKGROUND: The purpose of this study was to evaluate neointimal functionality of synthetic vascular grafts repopulated by host cells after implantation. METHODS: We obtained reseeded inflow and outflow cannulas of 2 patients undergoing orthotopic heart transplantation after left ventricular assist device implantation 9 and 10 months before. After cell isolation we examined cellular function of reseeded cells and their capability to form a functional endothelial layer applying immunohistologic markers and quantitative Western blot for endothelial nitric oxide synthase activity. RESULTS: Neointima formation in inflow and outflow cannulas differs macroscopically and by histologic appearance. The neointima formation on the surface of the polyethylene terephthalate fiber (Dacron) grafts differs substantially from native aortic vessel wall with respect to cellular and extracellular matrix composition and cellular function. CONCLUSIONS: The neointima of Dacron prostheses is composed of cells with rudimentary physiologic endothelial function. We conclude that synthetic matrices are not suitable scaffolds for generating functional cardiovascular implants.
Replacement of the trachea with an autologous aortic graft.
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Impact of multivessel coronary artery disease on outcome after isolated minimally invasive bypass grafting of the left anterior descending artery.
BACKGROUND: The outcome in patients treated surgically for coronary artery disease is known to be influenced by the extent of the disease. Whether this factor also has an effect in patients undergoing isolated minimally invasive revascularization of the left anterior descending (LAD) artery using the internal thoracic artery (ITA) (MIDCAB) has not been looked at. Thus, this study sought to evaluate the impact of multivessel disease (MVD) on midterm outcome after MIDCAB. METHODS: From 1996 to 1999, 411 patients received a MIDCAB at our institution and were now followed up. Isolated disease of the LAD (SVD -single vessel disease) was presented in 262 patients (63.7%) and 149 patients (36.3%) had MVD at the time of operation. The reasons for apparent incomplete revascularization in patients with MVD were very small target vessels (< 1.0-mm diameter), stenoses of less than 50%, distal localization of the stenoses, long-term patency after angioplasty, or an extensive risk for sternotomy and(or) cardiopulmonary bypass. The midterm outcome was evaluated by questionnaires sent to the patients and their physicians. RESULTS: The mean follow-up was 29.4 +/- 11.1 months. The incidence of myocardial infarction was significantly higher in MVD as compared to SVD patients (8.1% vs 1.9%, p = 0.04). Patients with MVD had significantly more subsequent percutaneous transluminal coronary angioplasty (10.7% vs 5.3%, p = 0.049) and a similar number of repeat surgical revascularizations as compared to SVD patients. Patients with MVD had a significantly higher total 3-year mortality as compared to SVD patients by Kaplan-Meier estimate (8.7% vs 3.1%, relative risk [RR] = 2.56, p = 0.011). The 3-year cardiac mortality was significantly higher in patients with MVD as compared to SVD (4.0% vs 0.4%, RR = 9.48, p = 0.0054). After adjustment of baseline characteristics by Cox regression analysis, the 3-year risk of cardiac death was significantly higher in the MVD groups (RR = 2.2, confidence interval [CI] 95%: 1.8 to 4.65, p = 0.029). CONCLUSIONS: Patients with isolated disease of the LAD appear to benefit from ITA grafting in the form of a MIDCAB procedure. Here, it should be an approach of choice. The results show that MVD is an independent risk factor for outcome in patients undergoing a MIDCAB procedure. Nevertheless, the midterm morbidity and mortality in MVD patients after a MIDCAB procedure where the LAD is the only target vessel for interventional or surgical treatment is acceptable despite a higher morbidity than in SVD patients.
Expansion of chondrocytes in a three-dimensional matrix for tracheal tissue engineering.
BACKGROUND: The generation of autologous tracheal implants by tissue-engineering techniques is a promising concept for otherwise untreatable patients. A functional cartilaginous backbone represents a prerequisite for any bioartificial tracheal graft. The aim of this study was to define suitable cell types and culture conditions for the generation of tracheal cartilage. METHODS: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot. RESULTS: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis. CONCLUSIONS: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering.
Impact of diabetes on outcome following isolated minimally invasive bypass grafting of the left anterior descending artery.
BACKGROUND: The outcome in patients treated by conventional coronary artery bypass grafting (CABG) for coronary artery disease is negatively influenced by the presence of diabetes. The relative effect of diabetes in patients undergoing isolated minimally invasive revascularization of the left anterior descending artery (LAD) using the internal thoracic artery (ITA) has as yet not specifically been looked at. Thus, this study sought to evaluate the impact of diabetes on mid-term outcome following minimally invasive coronary artery bypass grafting (MIDCAB). METHODS: From 1996 to 1999, 411 patients received a MIDCAB procedure at our institution and were now followed up. In this study population there were 63 diabetic patients (15.3%) and 348 nondiabetic patients (84.7%). Isolated proximal stenoses or an occlusion of the LAD were present in 262 patients (63.7%), whereas 149 (36.3%) had multi-vessel disease (MVD) at the time of the MIDCAB procedure. The clinical outcome was evaluated by questionnaires sent to the patients and their physicians. RESULTS: The mean follow-up was 29.4 +/- 11.1 months. The incidence of myocardial infarction was significantly higher in diabetics as compared to nondiabetics (9.5% vs 3.2%, p = 0.034). Diabetics and nondiabetics had similar rates of subsequent revascularization procedures during follow-up. Cumulative total survival of diabetic and nondiabetic patients was not statistically different. The 3-year cardiac mortality was however significantly higher in diabetic than in nondiabetic patients if MVD was initially present (Kaplan-Meier estimate: 10.7% vs 2.5%, relative risk [RR] = 5.5, p = 0.017 by log-rank test). The 3-year cardiac mortality in diabetic and nondiabetic patients with isolated disease of the LAD (single vessel disease [SVD]) was not significantly different. After adjustment of baseline characteristics by Cox regression analysis the 3-year risk of cardiac death was significantly higher in the diabetic group (RR = 1.82, CI 95%:1.2 to 3.3, p = 0.045). CONCLUSIONS: The results support diabetes to be an independent risk factor for outcome in patients with MVD undergoing a MIDCAB procedure in analogy to those undergoing CABG procedures. Diabetics with isolated disease of the LAD, however, benefit out of proportion from this treatment modality.
Guided tissue regeneration: porcine matrix does not transmit PERV.
OBJECTIVE: For cardiovascular tissue engineering, acellularized scaffolds of porcine matrices have been successfully used. However, the possibility of porcine endogenous retrovirus (PERV) transmission remains debatable. In this study, we investigated whether acellularized porcine vascular scaffolds cause cross-species transmission of PERV in a xenogenic model. METHODS: Porcine pulmonary arteries were acellularized and implanted into sheep in orthotopic position (n=6). Cardiopulmonary bypass support was used for all operations. Blood samples were collected regularly up to 6 months after the operation, and cellular components were tested for PERV infection by PCR and RT-PCR. Grafts were explanted 6 and 12 months after implantation. Tissue samples were characterized by histology and electron microscopy and tested for PERV sequences. RESULTS: All animals survived the procedure and follow up until explantation of the grafts. PERV DNA was detectable in acellularized scaffolds of porcine matrices. Acellular porcine pulmonary arteries scaffolds were repopulated in vivo by autologous cells of the host, leading to a vessel consisting of all cellular components of the vessel wall. No PERV sequences were detectable neither in all tested peripheral blood samples nor in tissue samples of in vivo recellularized grafts up to 6 months after implantation. Electron microscopy revealed no signs of graft infection by retrovirus. CONCLUSIONS: Guided tissue regeneration of acellularized vascular porcine matrix scaffolds leads to structured vessels up to one year without risk of PERV transmisson.
Clinical-pathologic conference in general thoracic surgery: pulmonary artery fibrohistiocytic tumor in a child.
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First human transplantation of a bioengineered airway tissue.
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Experimental generation of a tissue-engineered functional and vascularized trachea.
OBJECTIVE: We sought to grow in vitro functional smooth muscle cells, chondrocytes, and respiratory epithelium on a biologic, directly vascularized matrix as a scaffold for tracheal tissue engineering. METHODS: Ten- to 15-cm-long free jejunal segments with their own vascular pedicle were harvested and acellularized from donor pigs (n = 10) and used as a vascular matrix. Autologous costal chondrocytes, smooth muscle cells, and respiratory epithelium and endothelial progenitor cells were first cultured in vitro and then disseminated on the previously acellularized vascular matrix. Histologic, immunohistologic, molecular imaging, and Western blotting studies were then performed to assess cell viability. RESULTS: The endothelial progenitor cells re-endothelialized the matrix to such an extent that endothelial cell viability was uniformly documented through 2-(18F)-fluoro-2'-deoxyglucose positron emission tomography. This vascularized scaffold was seeded with functional (according to Western blot analysis) smooth muscle cells and successfully reseeded with viable ciliated respiratory epithelium. Chondrocyte growth and production of extracellular cartilaginous matrix was observed as soon as 2 weeks after their culture. CONCLUSIONS: The fundamental elements for a bioartificial trachea were successfully engineered in vitro in a direct vascularized 10- to 15-cm-long bioartificial matrix. Future experimental work will be directed to give them a 3-dimensional aspect and a biomechanical profile of a functioning trachea.
Expression of epidermal growth-factor receptor in lymphangiomatosis: a new therapeutic target?
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Bioartificial tracheal grafts: can tissue engineering keep its promise?
A huge variety of graft materials and transplantation approaches have been applied for decades in order to generate a clinically applicable tracheal substitute; so far, without success. Today, tissue engineering, the creation of man-made functional biological organs or tissue replacements from biodegradable carrier structures and autologous cells, may represent an alternative to the shortage of suitable grafts for reconstructive airway surgery. Partial success has been obtained by numerous groups following different concepts and strategies. In this article, tissue engineering approaches towards the bioartificial airway prosthesis are discussed, focusing primarily on recent developments in the field.
Influence of scaffold thickness and scaffold composition on bioartificial graft survival.
Biological scaffolds exhibit advantageous properties for tissue engineering of small diameter vessels. The influence of their extracellular matrix (ECM) components during in vivo repopulation is unknown. We implanted different xenogenic vascular matrices in a rat model to determine the influence of scaffold-thickness and ECM composition on in vivo repopulation. Decellularized ovine jugular vein (JV, n=42), carotid artery (CA, n=42) and aorta (AO, n=42) were implanted subcutaneously in the neck of adult male rats. Animals were sacrificed 2, 4 and 8 weeks after implantation. Cell and matrix morphology of explanted scaffolds were characterized by hematoxylin-eosin and pentachrome staining. Monoclonal anti-rat-CD31 was used to identify revascularization. Quantification of cell density was done by DNA-isolation. THICKNESS OF IMPLANTED XENOGENIC SCAFFOLDS VARIED ACCORDING TO THE MATERIAL USED (AO: 3.0-3.8mm; CA: 0.7-0.88mm; JV: 0.35-0.61mm). Immunohistology revealed complete repopulation of AO, CA, and JV scaffolds with endothelial cells and myofibroblasts within 2 weeks. After 8 weeks of implantation, AO scaffolds were completely covered by an endothelial monolayer and showed signs of a central matrix degeneration. JV scaffolds were completely degenerated at this stage. In contrast, CA scaffolds showed preserved ECM with a normal myofibroblast population and endothelial cell coverage.