Thorsten Walles

Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon

Patient prognosis in lung cancer largely depends on early diagnosis. The exhaled breath of patients may represent the ideal specimen for future lung cancer screening. However, the clinical applicability of current diagnostic sensor technologies based on signal pattern analysis remains incalculable due to their inability to identify a clear target. To test the robustness of the presence of a so far unknown volatile organic compound in the breath of patients with lung cancer, sniffer dogs were applied. Exhalation samples of 220 volunteers (healthy individuals, confirmed lung cancer or chronic obstructive pulmonary disease (COPD)) were presented to sniffer dogs following a rigid scientific protocol. Patient history, drug administration and clinicopathological data were analysed to identify potential bias or confounders. Lung cancer was identified with an overall sensitivity of 71% and a specificity of 93%. Lung cancer detection was independent from COPD and the presence of tobacco smoke and food odours. Logistic regression identified two drugs as potential confounders. It must be assumed that a robust and specific volatile organic compound (or pattern) is present in the breath of patients with lung cancer. Additional research efforts are required to overcome the current technical limitations of electronic sensor technologies to engineer a clinically applicable screening tool.

Generation and transplantation of an autologous vascularized bioartificial human tissue.

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.

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.

Results of valve-sparing aortic root reconstruction in 158 consecutive patients

BACKGROUND This study assesses the durability and clinical outcome of valve-sparing aortic root reconstruction using the reimplantation technique in a single center cohort. METHODS From July 1993 to July 2001, 158 patients underwent replacement of the ascending aorta with native valve reimplantation. Mean age of patients was 52 +/- 17 years (9 to 84 years), 103 were men (65%). Thirty-four patients (22%) suffered from Marfans syndrome. Aortic dissection Stanford type A was present in 29 patients (19%) (22 acute, 7 chronic), and concomitant partial or total arch replacement was necessary in 57 patients (36%). One or more additional procedures were performed in 28 patients (18%). Mean follow-up was 36 +/- 25 months (0.4 to 96 months). RESULTS Thirty-day mortality was 3.8% (6 patients), but only 2.2% in elective patients. Mean bypass time was 169 +/- 50 minutes (99 to 440 minutes), aortic cross-clamp time was 129 +/- 31 minutes (79 to 205 minutes). In patients undergoing arch replacement, circulatory arrest was 26 +/- 18 minutes (7 to 99 minutes). During follow-up, there were 5 (3.3%) cardiac-related late deaths. Grade of aortic insufficiency (AI) decreased from 2.3 +/- 1.1 (0 to 4) preoperatively to 0.23 +/- 0.44 (0 to 2) postoperatively (p < 0.0001). Six patients required aortic valve replacement, 4 of those due to progressive AI. Average grade of AI increased significantly to 0.42 +/- 0.61 (0 to 3) at latest evaluation (p = 0.002). Two patients experienced a transient ischemic attack within the first postoperative week. No further thromboembolic complications were noticed. All patients presented with a favorable exercise tolerance. CONCLUSIONS The aortic valve reimplantation technique achieves excellent clinical outcome with few complications even in complex pathologies. Lack of anticoagulation and favorable durability encourage wider and earlier use of this technique.

Phase II Trial of a Trimodality Regimen for Stage III Non-Small-Cell Lung Cancer Using Chemotherapy As Induction Treatment With Concurrent Hyperfractionated Chemoradiation With Carboplatin and Paclitaxel Followed by Subsequent Resection: A Single-Center Study

PURPOSE We started a phase II trial of induction chemotherapy and concurrent hyperfractionated chemoradiotherapy followed by either surgery or boost chemoradiotherapy in patients with advanced, stage III disease. The purpose is to achieve better survival in the surgery group with minimum morbidity and mortality. PATIENTS AND METHODS Patients treated from 1998 to 2002 with neoadjuvant chemoradiotherapy and surgical resection for stage III NSCLC were analyzed. The treatment consisted of four cycles of induction chemotherapy with carboplatin/paclitaxel followed by chemoradiotherapy with a reduced dose of carboplatin/paclitaxel and accelerated hyperfractionated radiotherapy with 1.5 Gy twice daily up to 45 Gy. After restaging, operable patients underwent thoracotomy. Inoperable patients received chemoradiotherapy up to 63 Gy. Study end points included resectability, pathologic response, and survival. Results One hundred twenty patients were enrolled; 25% patients had stage IIIA, 73% had stage IIIB, and 2% stage IV. After treatment, 47.5% had downstaging, 29.2% had stable disease, and 23.3% had progressive disease. Thirty patients (25%) were not eligible for operation because of progressive disease, stable disease, and/or functional deterioration with one treatment-related death. The 30-day mortality was 5% in patients who underwent operation. The 5-year survival rate for 120 patients was 21.7%, and it was 43.1% in patients with complete resection. In postoperative patients with stage N0 disease, 5-year survival was 53.3%; if stage N2 or N3 disease was still present, 5-year survival was 33.3%. CONCLUSION Staging and treatment with chemoradiotherapy and complete resection performed in experienced centers achieve acceptable morbidity and mortality.

Acellularized porcine heart valve scaffolds for heart valve tissue engineering and the risk of cross-species transmission of porcine endogenous retrovirus

OBJECTIVE Acellularized porcine heart valve scaffolds have been successfully used for heart valve tissue engineering, creating living functioning heart valve tissue. However, there is concern about the possibility of porcine endogenous retrovirus transmission. In this study we investigated whether acellularized porcine heart valve scaffold causes cross-species transmission of porcine endogenous retrovirus in a sheep model. METHODS Acellularized porcine pulmonary valve conduits (n = 3) and in vitro autologous repopulated porcine pulmonary valve conduits (n = 5) were implanted into sheep in the pulmonary valve position. Surgery was carried out with cardiopulmonary bypass support. The animals were killed 6 months after the operation. Blood samples were collected regularly up to 6 months after the operation and tested for porcine endogenous retrovirus by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. In addition, explanted tissue-engineered heart valves were tested for porcine endogenous retrovirus after 6 month in vivo. RESULTS Porcine endogenous retrovirus DNA was detectable in acellularized porcine heart valve tissue. However, 6 months after implantation of in vitro and in vivo repopulated acellularized porcine heart valve scaffolds, no porcine endogenous retrovirus sequences were detectable in heart valve tissue and peripheral blood. CONCLUSION Acellularized porcine matrix scaffolds used for creation of tissue-engineered heart valves do not transmit porcine endogenous retrovirus.

Valve-sparing aortic root reconstruction in patients with significant aortic insufficiency

BACKGROUND To assess the feasibility and outcome of the valve-sparing aortic root reimplantation technique in patients with severe preoperative aortic insufficiency (AI). METHODS Within 8 years we have operated on 158 patients with aneurysms of the ascending aorta using the reimplantation technique. We identified 83 patients with AI grade 3 or 4 (mean 3.1 +/- 0.4) preoperatively (study group). This cohort was compared with 71 patients with AI grade 2 or less (mean 1.3 +/- 0.9; control group) with regard to mortality, operative variables, complications, need for reoperation, postoperative AI, and clinical presentation during follow-up. RESULTS Patient demographics were comparable in both groups. However, Marfans syndrome (32% versus 13%, p = 0.006) and acute type A aortic dissection (20% versus 8.4%, p = 0.059) were more frequent in the control group. In addition, bypass (177 +/- 60 minutes versus 160 +/- 36 minutes, p = 0.022) and cross clamp times (133 +/- 34 minutes versus 124 +/- 27 minutes, p = 0.049) were significantly longer in controls. Mortality was low in the study group and comparable with controls (30-day, 3.6% versus 4.2%; during follow-up, 3.8% versus 5.9%; p = not significant [NS]). Reoperation rate was almost identical in both groups (3.8% versus 4.4%, p = NS). Mean grade of AI was significantly higher in the study group early postoperatively (0.31 +/- 0.46 versus 0.18 +/- 0.42, p = 0.049) but comparable at the last visit (0.43 +/- 0.58 versus 0.42 +/- 0.62, p = NS). During follow-up neither thromboembolic complications nor bleeding events were noted in either group. Clinical performance at the last visit revealed no significant difference between the groups. CONCLUSIONS Preoperative severe aortic insufficiency does not impair the excellent outcome seen after a mean of 3 years of follow-up in patients undergoing the reimplantation technique for valve-sparing aortic root reconstruction.

Engineering of fibrillar decorin matrices for a tissue-engineered trachea

Decorin is a structural and functional proteoglycan (PG) residing in the complex network of extracellular matrix (ECM) proteins in many connective tissues. Depending on the protein core and the glycosaminoglycan chain, PGs support cell adhesion, migration, proliferation, differentiation, ECM assembly and growth factor binding. For applications in tissue engineering, it is crucial to develop reliable, ECM-mimicking biomaterials. Electrospinning is a suitable method for creating three-dimensional (3D), fibrillar scaffolds. While there are numerous reports on the electrospinning of proteins including collagen, to date, there are no reports on the electrospinning of PGs. In the following study, we used electrospinning to generate decorin-containing matrices for tracheal tissue engineering applications. The electrospun scaffolds were analyzed using scanning electron microscopy, atomic force microscopy, contact angle measurements and dynamic mechanical analysis. Additionally, we confirmed PG functionality with immunostaining and 1,9-dimethylmethylene blue. To determine cell-matrix-interactions, tracheal cells (hPAECs) were seeded and analyzed using an FOXJ1-antibody. Moreover, interactions of the electrospun scaffolds with immune-mediated mechanisms were analyzed in detail. To conclude, we demonstrated the feasibility of electrospinning of decorin to generate functional 3D scaffolds with low immunogenicity for hPAEC expansion. Our data suggest that these hybrid materials may be suitable as a substrate for tracheal tissue engineering.

Establishment of a human 3D lung cancer model based on a biological tissue matrix combined with a Boolean in silico model

For the development of new treatment strategies against cancer, understanding signaling networks and their changes upon drug response is a promising approach to identify new drug targets and biomarker profiles. Pre‐requisites are tumor models with multiple read‐out options that accurately reflect the clinical situation. Tissue engineering technologies offer the integration of components of the tumor microenvironment which are known to impair drug response of cancer cells. We established three‐dimensional (3D) lung carcinoma models on a decellularized tissue matrix, providing a complex microenvironment for cell growth. For model generation, we used two cell lines with (HCC827) or without (A549) an activating mutation of the epidermal growth factor receptor (EGFR), exhibiting different sensitivities to the EGFR inhibitor gefitinib. EGFR activation in HCC827 was inhibited by gefitinib, resulting in a significant reduction of proliferation (Ki‐67 proliferation index) and in the induction of apoptosis (TUNEL staining, M30‐ELISA). No significant effect was observed in conventional cell culture. Results from the 3D model correlated with the results of an in silico model that integrates the EGFR signaling network according to clinical data. The application of TGFβ1 induced tumor cell invasion, accompanied by epithelial–mesenchymal transition (EMT) both in vitro and in silico. This was confirmed in the 3D model by acquisition of mesenchymal cell morphology and modified expression of fibronectin, E‐cadherin, β‐catenin and mucin‐1. Quantitative read‐outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model. The combined in vitro and in silico model represents a powerful tool for systems analysis.

Guided Tissue Regeneration of Vascular Grafts in the Peritoneal Cavity

To the Editor: Tissue engineering represents an upcoming alternative source for vascular substitutes to create viable and biologically active grafts. Two different concepts are followed: grafts are either reseeded in vitro before implantation (tissue engineering)1 or the scaffolds are implanted as acellular matrices for intrinsic reseeding in vivo (guided tissue regeneration).2,3⇓ The scaffold matrices are fashioned from natural materials or synthetic polymers.4,5⇓ Despite considerable clinical research, no biological or synthetic grafts have been produced so far as an ideal substitute for a small-diameter artery.5,6⇓ Recently, our group focused research on the creation of bioartificial blood vessel grafts. Therefore, we read with interest the study in Circulation Research by Campbell et al7 …