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Suggesting that bioartificial vascular scaffolds cannot but tissue-engineered vessels can withstand biomechanical stress, we developed in vitro methods for preclinical biological material testings. The aim of the study was to evaluate the influence of revitalization of xenogenous scaffolds on biomechanical stability of tissue-engineered vessels. For measurement of radial distensibility, a salt-solution inflation method was used. The longitudinal tensile strength test (DIN 50145) was applied on bone-shaped specimen: tensile/tear strength (SigmaB/R), elongation at maximum yield stress/rupture (DeltaB/R), and modulus of elasticity were determined of native (NAs; n = 6), decellularized (DAs; n = 6), and decellularized carotid arteries reseeded with human vascular smooth muscle cells and human vascular endothelial cells (RAs; n = 7). Radial distensibility of DAs was significantly lower (113) than for NAs (135) (P < 0.001) or RAs (127) (P = 0.018). At levels of 120 mm Hg and more, decellularized matrices burst (120, 160 n = 2 and 200 mm Hg). Although RAs withstood levels up to 300 mm Hg, ANOVA revealed a significant difference from NA (P = 0.018). Compared with native vessels (NAs), SigmaB/R values were lower in DAs (44%; 57%) (P = 0.014 and P = 0.002, respectively) and were significantly higher in RAs (71%; 83%) (both P < 0.001). Similarly, DeltaB/R values were much higher in DAs compared with NAs (94%; 88%) (P < 0.001) and RAs (87%; 103%) (P < 0.001), but equivalent in NAs and RAs. Modulus of elasticity (2.6/1.1/3.7 to 16.6 N/mm(2)) of NAs, DAs, RAs was comparable (P = 0.088). Using newly developed in vitro methods for small-caliber vascular graft testing, this study proved that revitalization of decellularized connective tissue scaffolds led to vascular graft stability able to withstand biomechanical stress mimicking the human circulation. This tissue engineering approach provides a sufficiently stable autologized graft.

Objectives: Tissue-engineered arterial vessels (TEAV) offer substantial advantages in small-calibered human-bypass- grafting and vascularized scaffold applications. However, histological composition of TEAV must allow for functional properties, such as vasomotoricity. Aim of this study was to characterize human TEAVs regarding morphology and vasomotoricity. Methods: Three groups containing segments of porcine carotid artery < 5mm in diameter (native [NA, n = 6], decellularized [DA, n = 6], and decellularized/reseeded in a bioreactor [RA, n 7] with human vascular endothelial [hvECs] and smooth muscle cells [hvSMCs]) were examined. Light and scanning electron microscopy were applied, and hvSMCs- and hvECs-associated Vasomotoricity Test conducted in Krebs-solution was used for characterization of revitalized TEAVs. Results: Morphologic examination showed cell-free extracellular matrix in DAs. Light microscopy demonstrated intact extracellular matrix components in circle-layered formation in cross sections of DAs. RAs showed small cells migrating along the remaining medial fiber structures and flat cell layers at the luminal site, identified as hvECs and hvSMCs with lower CD-31 and alpha-actin signaling than controls. Scanning electron microscopy showed intact flat cell layers on luminal surfaces of RAs and dense hvSMCs at their media site. DAs showed decreasing strain after stimulation. RAs retrieved vasomotoricity compared to DAs, but showed reduced contraction and incomplete relaxation compared to NAs. Conclusions: This study shows that revitalization of DA with human vascular cells resembles NA-like morphology and can ensure vasomotoricity of TEAVs.

BackgroundTissue-engineered pulmonary arteries could overcome the drawbacks of homografts or prosthetic conduits used in the repair of many congenital cardiac defects. However, the ideal scaffold material for tissue-engineered conduits is still subject of intensive debate. In this study, we evaluated an acellularized allogeneic matrix scaffold for pulmonary artery tissue engineering with and without in-vitro reseeding with autologous endothelial cells in the pulmonary circulation in a growing sheep model.MethodsOvine pulmonary arteries (n = 10) were acellularized by trypsin/ethylenediamine tetraacetic acid incubation. Autologous endothelial cells were harvested from carotid arteries, and the pulmonary conduits were seeded with endothelial cells. We implanted in-vitro, autologous, reendothelialized (group A, n = 5) and acellularized pulmonary conduits (group B, n = 5) in the pulmonary circulation. The animals were sacrificed 6 months after the operation. Explanted valves were examined histologically and by immunohistochemistry.ResultsThe conduit diameter increased in both groups (group A, 44% ± 11%; group B, 87% ± 18%; p < 0.05). In group A, however, a proportional increase in diameter was present, whereas in group B, a disproportionate increase resulting in aneurysm formation was observed. Histologically, the conduit wall integrity was destroyed in group B and preserved in group A. In group B, the extracellularmatrix degenerated with a reduced amount of collagens and proteoglycanes. Furthermore, no elastic fibers were detectable. In contrast, the extracellularmatrix in group A was close to native ovine tissue.ConclusionsTissue-engineered pulmonary conduits (autologous endothelial cells and allogeneic matrix scaffolds) functioned well in the pulmonary circulation. They demonstrated an increase in diameter and an extracellular matrix comparable to that of native ovine tissue.

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.

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.

BACKGROUND The ideal scaffold material for tissue engineered heart valves is discussed controversially. We evaluated acellularized xenogenic matrix constructs with and without seeding with autologous vascular cells in the pulmonary circulation in a sheep model. METHODS Porcine pulmonary valve conduits (n=16) were acellularized by trypsin/ EDTA incubation. Autologous myofibroblasts and endothelial cells were harvested from carotid arteries; xenogenic valve conduits (n=10) were repopulated with these autologous cells resulting in uniform cellular restitution of the pulmonary valve conduit surface. Using this method, we implanted autologous cell/xenogenic matrix constructs (XB) in ten animals. In six control animals acellularized/xenogenic matrix constructs (XA) were implanted. In each animal, cardiopulmonary bypass was used to resect the pulmonary valve and replace it with the xenogenic pulmonary valve conduits. The animals were killed after 6, 9 or 12 months. The explanted valves were examined histologically and biochemically. RESULTS After explantation XB showed severe cusp degeneration, which resulted in severe valvular regurgitation. In comparison, XA appeared macroscopically normal with preserved valvular function. The surface of XB were covered with an incomplete endothelial multilayer. The extracellular matrix (ECM) of XB showed pathological amounts of collagenous and elastic fibers as well as proteoglycan content combined with an increase cellularity. The XA were completely repopulated by an endothelial cell monolayer; the ECM was repopulated with a myofibroblast population comparable to native ovine heart valve tissue. CONCLUSIONS Approaches to heart valve engineering based on acellularized/xenogenic matrices provide promising results and will hopefully led to the "ideal" valve substitute in clinical heart valve replacement.

Background: Partial or radical cystectomy requires replacement of the urinary reservoir normally achieved by using small or large bowel segments. Our aim was to establish tissue engineering of an bioartificial bladder wall using primary cultures of porcine urothelial (pUC) and bladder smooth muscle cells (pSMC) to be reseeded on different acellular biological matrices. Methods: Primary porcine cultures of pUC and pSMC were established from open bladder biopsy material 25 mm 2 in size. Acellular matrix was generated either from a) porcine bladder wall segments or b) tubular small intestinal submucosa with the still attached decellularized muscularis layer. Reseeding of these matrices with primary cells was done in a two-dimensional static model and in a three-dimensional rotating bioreactor perfused with cell culture medium for a period of 6 weeks. Results: Prior to reseeding the cultured cells were characterized as pUC and pSMC by immunohistochemical staining with either anti-keratin 7 or anti-alpha actin. For both matrices a reseeded double layer cell system of pUC and pSMC could be identified after incubation in the described systems for 6 weeks. Conclusions: Our results document successful generation of tissue engineered urinary bladder wall, which can be used in further large animal transplantation experiments.

BACKGROUND: Internal thoracic arterial grafts (ITA) in coronary artery bypass surgery provide excellent long-term patency results. Due to the elevated incidence of sternal infections following pedicled ITA harvesting, blood supply to the sternum has gained the focus of attention. This study sought to evaluate real time parameters of sternal microcirculation prior and immediately after harvesting of the ITA by a novel laser Doppler flowmetry and remission spectroscopy system (Oxygen-To-See (O2C), LEA Medizintechnik, Giessen). METHODS: 21 patients (16 males, age 63 + 4 years, mean NYHA 2.3 +/- 0.3) scheduled for coronary artery bypass grafting (CABG) were enrolled into the study. After median sternotomy, the probe was placed sequentially pre- and retrosternally for measurements of tissue oxygen saturation (sO2), hemoglobin concentration (rHb), superficial (2 mm) und deep (8 mm) blood flow. Measurements were performed and analyzed before and after surgical harvesting of the ITA with a pedicle. RESULTS: Baseline pre- and retrosternal tissue oxygen saturation (sO2) were 90 +/- 3 % and 87 + 4 %, respectively (n. s.). After left ITA harvesting, presternal sO2 remained unchanged (90 + 4 %, n. s.), whereas retrosternal sO2 decreased significantly (54 + 4 %, p < 0.001). Simultaneously, retrosternal post-capillary venous filling (rHb) increased significantly after ITA harvesting (86 +/- 2 vs. 93 + 2, p < 0.05), whereas presternal rHb remained unchanged. Retrosternal superficial and deep blood flow also decreased significantly (75 +/- 5 vs. 41 +/- 4, and 94 +/- 5 vs. 52 +/- 6) in contrast to comparable presternal blood flow before and after ITA harvesting. There were neither superficial nor deep sternal wound infections occurred in the studied patient population. CONCLUSIONS: The pedicled harvesting of ITA leads to a significant decrease of microcirculatory blood flow, retrosternal tissue oxygen saturation, and an increase in post-capillary venous filling. Parameters of microcirculation in the presternal area after ITA harvesting remained unchanged compared to baseline values. Hence, the incidence of sternal infections after ITA harvesting in coronary surgery may well be explained by a significant decrease of sternal blood supply in the retrosternal area. Further prospective randomized studies are needed to elucidate the potential role of skeletonized ITA preparation in sternal microcirculation.

Various types of three-dimensional matrices have been used as basic scaffolds in myocardial tissue engineering. Many of those are limited by insufficient mechanical function, availability, or biocompatibility. We present a clinically established collagen scaffold for the development of bioartificial myocardial tissue. Neonatal rat cardiomyocytes were seeded into Tissue Fleece (Baxter Deutschland, Heidelberg, Germany). Histological and ultrastructural examinations were performed by DAPI and DiOC(18) staining and electron microscopy, respectively. Force measurements from the spontaneously beating construct were obtained. The constructs were stimulated with agents such as adrenalin and calcium, and by stretching. Passive stretch curves were obtained. Spontaneous contractions of solid bioartificial myocardial tissue (BMT), 20 x 15 x 2 mm, resulted. Contractions continued to week 12 (40% of BMTs) in culture. Histology revealed intercellular and also cell-fibril junctions. Elasticity was similar to that of native rat myocardium. Contractile force increased after topical administration of Ca(2+) and adrenaline. Stretch led to the highest levels of contractile force. In summary, bioartificial myocardial tissue with significant in vitro longevity, spontaneous contractility, and homogeneous cell distribution was produced using Tissue Fleece. Tissue Fleece constitutes an effective scaffold to engineer solid organ structures, which could be used for repair of congenital defects or replacement of diseased tissue.

Background Approaches to in vivo repopulation of acellularized valve matrix constructs have been described recently. However, early calcification of acellularized matrices repopulated in vivo remains a major obstacle. We hypothesised that the matrix composition has a significant influence on the onset of early calcification. Therefore, we evaluated the calcification of acellularized allogenic ovine (AVMC) and xenogenic porcine (XVMC) valve matrix conduits in the pulmonary circulation in a sheep model. Methods Porcine (n = 3) and sheep (n = 3) pulmonary valve conduits were acellularized by trypsin/EDTA digestion and then implanted into healthy sheep in pulmonary valve position using extracorporeal bypass support. Transthoracic echocardiography (TTE) was performed at 12 and 24 weeks after the implantation. The animals were sacrificed at week 24 or earlier when severe calcification of the valve conduit became evident by TTE. The valves were examined histologically and biochemically. Results All AVMC revealed severe calcification after 12 weeks with focal endothelial cell clustering and no interstitial valve tissue reconstitution. In contrast, after 24 weeks XVMC indicated mild calcification on histologic examination (von Kossa staining) with histologic reconstitution of valve tissue and confluent endothelial surface coverage. Furthermore, immunohistologic analysis revealed reconstitution of surface endothelial cell monolayer (von Willebrand factor), and interstitial myofibroblasts (Vimentin/Desmin). Conclusions Porcine acellularized XVMC are resistant to early calcification during in vivo reseeding. Furthermore, XVMC are repopulated in vivo with valve-specific cell types within 24 weeks resembling native valve tissue.

Objective Degradation mechanisms of cardiovascular bioprostheses may play an important role in bioartificial implants. The fate of acellular implanted and cellular cardiovascular scaffolds was examined in an in vivo model. Methods Decellularized or native ovine carotid artery (CA, n=42) and aorta (AO, n=42) were implanted subcutaneously into rats for 2, 4 and 8 weeks. Immunohistochemical methods were used to monitor repopulation. Desmin-vimentin, CD31-, CD4- and CD18-antibodies for myocytes, endothelium, and inflammatory cell-infiltration, respectively. Calcification was detected by von-Kossa staining. Cell density was quantified by DNA-isolation. Results Acellular AO and CA matrices showed progressive calcification. Cellular AO and CA matrices trigger a strong inflammatory reaction which subsides after two weeks. CA scaffolds are revascularized progressively, whereas AO biocomposites degenerate. Calcification is less pronounced in cellular AO scaffolds and lacking in CA. Conclusions Acellular bioartificial implants demonstrate degradation mechanisms similar to currently applied cardiovascular bioprostheses. Cellularized viable implants are promising clinical alternatives.

OBJECTIVES: After a period of 5 to 10 years, biological heart valve prostheses undergo degenerative processes, which finally lead to dysfunction and complete destruction. Although many efforts have been made to identify underlying mechanisms, many questions remain unanswered. Here we evaluate immunological factors and their potential role in biological heart valve destruction. PATIENTS AND METHODS: Allogeneic (n=10) and xenogeneic (n=3) aortic valvular prostheses, as well as aortic valves retrieved from transplanted human hearts, which had to be replaced because of chronic graft rejection (n=4) were analyzed. Aortic valves from human donor hearts (native) (n=4), which were considered not transplantable served as controls. Endothelial expression patterns of the following adhesion molecules were analyzed by immunohistochemistry: selectin family: ELAM-1, CD62, integrin family: VLA-1, -2, -3, -4, -5, and -6, immunoglobulin supergene family: PECAM-1, ICAM-1, and -2, and class I heavy chain proteins, complementary adhesion molecules: CD34, CD44 and the von Willebrand factor. RESULTS: ELAM-1, ICAM-1 and -2, CD34, CD44 and class I heavy chain proteins, all molecules which play significant roles during inflammatory processes, showed stronger expression patterns in allogeneic and xenogeneic aortic heart valve prostheses compared to native or chronically rejected valves. Furthermore, the von Willebrand factor stained positive only on allogeneic and xenogeneic valves. Only mild differences were observed regarding the expression of integrin molecules and CD62. CONCLUSIONS: Immunological reactions play a major role in the degeneration of biological heart valve prostheses. This is underlined by observations made on aortic valves from chronically rejected cardiac grafts, which did not show any degenerative alterations. Thus, since immunosuppressive therapy after heart valve replacement is no reasonable alternative, novel and future approaches in "tissue engineering" will hopefully help avoid tissue degeneration, while preserving the advantage of biological tissue origin.

OBJECTIVES Endothelial cells play a significant role in cardiovascular physiology and the pathogenesis of numerous cardiovascular diseases. Essential phenomena such as hemostasis, inflammation and immunity require close interactions between immunocompetent and endothelial cells. However, many questions of endothelial heterogeneity regarding function and morphology at various vascular sites remain unanswered. In this study, we have created an immunohistochemical map of endothelial adhesion molecule expression at different vascular sites of the healthy human heart. The main focus was to analyze endothelial expression patterns and whether they are distinctive regarding their function at these vascular sites. We also tried to build up a relation between clinical and immunohistochemical findings. PATIENTS AND METHODS Tissue samples from eleven different vascular locations of healthy human hearts were analyzed using immunohistochemistry. Endothelial adhesion molecules of the selectin, immunoglobulin supergene, and integrin families, some complementary cellular adhesion molecules, and von Willebrand factor were analyzed. RESULTS Endothelial adhesion molecule expression was found to be characteristic for all vascular sites investigated. Thus, molecules involved in inflammatory reactions were predominantly expressed within the myocardial microvasculature, whereas molecules serving endothelial anchorage towards extracellular matrix components could be observed especially on endocardial and valvular surfaces. Apart from that, a parallel expression of immunologically relevant as well as integrin molecules was found to be characteristic for coronary arteries. CONCLUSIONS To our knowledge, this is the first report on site-specific expression characteristics for all vascular sites of the human heart. Thus, our data provide important novel information, which will ultimately help to bring some light into the field of cardiac physiology.

Objective: Replacement of heart valves in human subjects has become a routine procedure in cardiac operations. We sought to investigate whether commercially available glutaraldehyde-fixed porcine heart valve prostheses cause porcine endogenous retrovirus infection in human subjects because recent studies revealed that human cells can be infected with porcine endogenous retrovirus. Methods: Blood samples of 18 patients who underwent aortic or mitral valve replacement with porcine heart valves were collected 6 months to 3 years after operation and tested for porcine endogenous retrovirus by means of polymerase chain reaction and reverse transcriptase–polymerase chain reaction. In addition, we tried to trace porcine endogenous retrovirus in 3 commercially available, glutaraldehyde-fixed, porcine heart valves. Results: Porcine endogenous retrovirus can be easily detected in native porcine heart valves and degrades completely within 1 week of fixation in glutaraldehyde. In all 3 commercially available porcine heart valves, no traces of porcine endogenous retrovirus were found. All blood samples showed negative test results for the porcine endogenous retrovirus genome. Conclusion: Our results indicate that glutaraldehyde fixation of porcine heart valves reliably prevents cross-species transmission of porcine endogenous retrovirus. (J Thorac Cardiovasc Surg 2001;121:697-701)

The 3-D skin equivalent can be viewed as physiologically comparable to the natural skin and therefore is a suitable alternative for animal testing. This highly differentiated in vitro human skin equivalent is used to assess the efficacy and mode of action of novel agents. This model is generated from primary human keratinocytes on a collagen substrate containing human dermal fibroblasts. It is grown at the air-liquid interface which allows full epidermal stratification and epidermal-dermal interactions to occur. Future emphasis is the establishment of different test systems to investigate wound healing, melanoma research and infection biology. Key features of this skin model are that it can be used as an alternative for in vivo studies, donor tissue can be tailored to the needs of the study and multiple analyses can be carried out at mRNA and protein level. Driven by both ethical and economical incentives, this has already resulted in a shift of the test strategies used by the Pharmaceutical Industry in the early drug development process as reflected by the increased demand for application of cell based assays. It is also a suitable model for testing a wide variety of endpoints including cell viability, the release of proinflammatory mediators, permeation rate, proliferation and biochemical changes.

Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After filtration, identification is carried out on the single particle level: image processing detects possible contaminants and prepares them for Raman spectroscopic analysis. A custom-built Raman-spectrometer-attachment coupled to the commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step, the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and classification is carried out based on a support vector machine.

The objective of the study was to describe a novel small-animal model of tissue-engineered aortic valve conduits and to investigate biological processes in an accelerated and inexpensive fashion. An isogenic Lewis-to-Lewis rat model was used to exclude immunological factors of graft deterioration. U-shaped aortic valvular grafts were decellularized and characterized morphologically. Acellular conduits were repopulated with labeled isogenic cells in a bioreactor under flow conditions. Grafts were anastomosed to the recipient's abdominal aorta in an end-to-side manner (n = 7). Native rat aortas were implanted as a control group (n = 7). Grafts were explanted after 28 days and characterized. After treatment with trypsin-ethylenediaminetetraacetic acid, no residual cells were visualized in the scaffold. Mean DNA content decreased from 0.347 to 0 microg/mg of DNA/tissue, and the content of collagenous connective tissue and proteoglycans appeared slightly reduced. Isolated aortic rat endothelial cells and myofibroblasts were repopulated on the acellularized scaffold, and fluorescent-labeled myofibroblasts were identified in the meshwork. Endothelial cells formed a monolayer on the luminal surface. Reseeded cells were viable as ascertained using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. After implantation, Doppler and M-mode echography proved pulsatile cusp movement. All conduits were patent after 28 days. Examination of tissue-engineered explants revealed thickened aortic walls and incompetent valve function. Microscopically, aortic intima and media appeared normal, whereas the adventitia showed hyperproliferation of fibroblasts. Our new model leads to accelerated and reproducible results, suited to investigation of biological patterns of tissue engineering. The observed adventitial fibrosis emphasized the importance of careful selection of optimal cell types for repopulation in tissue-engineered constructs.

Background: Porcine endogenous retrovirus (PERV) remains a safety risk in pig-to-human xenotransplantation. There is no evidence of in vivo productive infection in humans because PERV is inactivated by human serum. However, PERV can infect human cell lines and human primary cells in vitro and inhibit human immune functions. Aims: We investigated the potential of primary porcine liver cells to transmit PERV to primary human cells in a bioreactor-based bioartificial liver (BAL). Methods: Primary human hepatocytes, endothelial cells and the human cell line HEK 293 were exposed to supernatants from BAL or from the porcine cell line PK-15. PERV polymerase-specific reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR were used to investigate PERV transmission to human cells. An assay of RT activity was used to detect the presence of retrovirus in the supernatants of BAL, primary human hepatocytes and endothelial cells. Results: Primary human hepatocytes (hHep), endothelial cells and HEK 293 cells were reproducibly infected by PERV, originating from primary porcine liver cells within the BAL and from PK-15 cells. Infected cells were positive for PERV-specific DNA and RNA after 8–10 days on an average, and RT activity was detectable in the supernatants of infected hHep and HEK 293 cells. Conclusion: A risk of PERV infection in human cells is documented in this study, indicating that short-term contact of primary porcine liver cell supernatants with primary human cells could result in PERV transmission.

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 all 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 ill vitro. Clinical tissue transplantation confirmed vessel patency and tissue viability for 1 week. Conclusions. The feasibility to bioengineer a human tissue with all 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.

To gain basic insight into the impact of non-biological features on cells’ behaviour, primary skin-cells, keratinocytes and fibroblasts, were cultured on amine-functionalized or carboxy-functionalized planar, nano- or microstructured surfaces. Sintered layers of silica nano- or microparticles were used to fabricate structures in the range of naturally occurring structure-sizes. Organo-chemical functionalization was achieved using organo-functional silanes. Primary human keratinocytes and fibroblasts were isolated from human foreskin and cultivated on the modified interfaces. Both cell-types displayed specific proliferation behaviour, depending on surface topography and chemical functionalization: Keratinocytes showed significantly better proliferation on amino-functionalized surfaces than on carboxy-functionalized surfaces. On amino-functional surfaces decree-topography. Fibroblasts, in contrast, tended to proliferate stronger on carboxylated surfaces. Immunohistological staining proofed that actin and vinculin, which is involved in the formation of focal adhesions, were expressed on all modified surfaces, thus revealing intact cytoskeleton and cell-substrate contacts.

Sterility testing of cell or tissue cultures is an essential task in the fields of regenerative medicine and tissue engineering. Especially in case of Good manufacturing practice (GMP) of cell and tissue based transplants. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis. A Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. Identification of critical particles like microorganisms via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. However an automated image analysis of small particles from supernatant of biopsies on a filter chip with tiny holes is a difficult task. Especially for the discrimination of small particles like cell debris and bacteria, which have a quite similar range of size. Because of that particles in the supernatant and microorganisms have to be discriminated by means of Raman spectroscopy. We present here a Raman based method to discriminate between cells, microorganisms and particles in cell culture.

Die moderne Zell- und Molekularbiologie ist ohne den Einsatz von Zell- und Gewebekulturen nicht denkbar. Die „In-vitro-Zellbiologie“ bildet heute die Grundlage fur die Aufklarung grundlegender Wachstums- und Differenzierungsprozesse tierischer Zellen. Saugerzellkulturen finden Einsatz in der biomedizinischen Grundlagenforschung, der Virologie, Immunologie, Strahlenbiologie und Krebsforschung. Daruber hinaus spielen Zellkulturen eine grose Rolle in der medizinischen Diagnostik (u. a. pranatale Diagnose, Tumordiagnostik), zur Herstellung von biologischen Wirkstoffen fur Diagnostik, Prophylaxe und Therapie, zur Produktion von Impfstoffen und Arzneimitteln und fur toxikologische Untersuchungen. Durch neueste Entwicklungen auf den Gebieten der Stammzellforschung und des Tissue Engineering werden Zellkulturen mehr und mehr Eingang in die regenerative Medizin finden.

Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the critical particles are automatically baseline-corrected without using background data and classified via hierarchical cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.

Liver tissue that is functional and viable for several weeks in vitro represents an auspicious test system for basic and applied research. In this study, a coculture system for hepatocytes (HCs) and microvascular endothelial cells (mECs) was generated applying tissue-engineering techniques, establishing the basis for a new bioartificial liver in vitro model. Porcine mECs were seeded on a decellularized porcine jejunal segment with preserved vascular structures. Porcine HCs were seeded onto this vascularized scaffold, and the resulting coculture was maintained for 3 weeks in vitro. Tissue morphology and differentiation was monitored using histology and immunohistochemistry. Tissue metabolism was monitored using daily assessment of urea and lactate production. HC monolayer cultures served as controls. The 2-stage seeding procedure resulted in a 3-dimensional coculture system harbouring HC cell clusters in multiple cell layers lining the generated mEC-seeded capillary structures. It was viable for 3 weeks, and HCs maintained their morphology and differentiation. Biochemical testing revealed stable metabolic activity of the tissue culture. In contrast, HCs cultured in monolayer showed morphological dedifferentiation and an unfavorable metabolic state. Our mEC-HC coculture represents a new approach toward a functional bioartificial liver-like tissue applicable as a test system for basic and applied research.

This review article addresses the relevance and potential of bioartificial tissues in oncologic research and therapy and reconstructive oncologic surgery. In order to translate the findings from basic cellular research into clinical applications, cell-based models need to recapitulate both the 3D organization and multicellular complexity of an organ but at the same time accommodate systematic experimental intervention. Here, tissue engineering, the generation of human tissues and organs in vitro, provides new perspectives for basic and applied research by offering 3D tissue cultures resolving fundamental obstacles encountered in currently applied 2D and 3D cell culture systems. Tissue engineering has already been applied to create replacement structures for reconstructive surgery. Applied in vitro, these complex multicellular 3D tissue cultures mimic the microenvironment of human tissues. In contrast to the currently available cell culture systems providing only limited insight into the complex interactions in tissue differentiation, carcinogenesis, angiogenesis and the stromal reaction, the more realistic (micro) environment afforded by the bioartificial tissue-specific 3D test systems may accelerate the progress in design and development of cancer therapies.

Background— Tissue engineering (TE) of heart valves reseeded with autologous cells has been successfully performed in vitro. Here, we report our first clinical implantation of pulmonary heart valves (PV) engineered with autologous endothelial progenitor cells (EPCs) and the results of 3.5 years of follow-up. Methods and Results— Human PV allografts were decellularized (Trypsin/EDTA) and resulting scaffolds reseeded with peripheral mononuclear cells isolated from human blood. Positive stain for von Willebrand factor, CD31, and Flk-1 was observed in monolayers of cells cultivated and differentiated on the luminal surface of the scaffolds in a dynamic bioreactor system for up to 21 days, indicating endothelial nature. PV reseeded with autologous cells were implanted into 2 pediatric patients (age 13 and 11) with congenital PV failure. Postoperatively, a mild pulmonary regurgitation was documented in both children. Based on regular echocardiographic investigations, hemodynamic parameters and cardiac morphology changed in 3.5 years as follows: increase of the PV annulus diameter (18 to 22.5 mm and 22 to 26 mm, respectively), decrease of valve regurgitation (trivial/mild and trivial, respectively), decrease (16 to 9 mm Hg) or a increase (8 to 9.5 mm Hg) of mean transvalvular gradient, remained 26 mm or decreased (32 to 28 mm) right-ventricular end-diastolic diameter. The body surface area increased (1.07 to 1.42 m 2 and 1.07 to 1.46 m 2 , respectively). No signs of valve degeneration were observed in both patients. Conclusions— TE of human heart valves using autologous EPC is a feasible and safe method for pulmonary valve replacement. TE valves have the potential to remodel and grow accordingly to the somatic growth of the child.

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.

OBJECTIVE To investigate the influence of commercially available vasoactive drugs on human cavernosal endothelial and fibroblastic cells in vitro, as although corporal fibrosis is a well known side-effect of intracavernosal injection therapy for erectile dysfunction, the possible detrimental effect of these agents on the endothelium lining the cavernosal vascular spaces is uncertain. MATERIALS AND METHODS Cultured primary endothelial (13) and fibroblastic cells (12), obtained from potent patients undergoing penile surgery, were exposed to different physiological dilutions of prostaglandin E1 (PGE1), papaverine/phentolamine or the respective triple-mix of these agents for 30 min. Viable cells were counted and cell metabolic activity measured in these cultures 48 h after drug exposure. RESULTS There was a significant dose-dependent decrease in the viable cell count after exposure to papaverine-containing formulations, probably because of the low pH of this substance. This cytotoxic effect was more pronounced in endothelial than in fibroblastic cells, and was not apparent in the PGE1 groups. The relative increase in cell metabolic activity in cultures affected by a moderate cytotoxic effect indicated a regenerative process. CONCLUSION These comparative results in endothelial and fibroblastic cell cultures suggest that the endothelium rather than the interstitium of the corpus cavernosum is more sensitive to side-effects produced by intracavernosal injection therapy with papaverine. Thus, unfavourable consequences on the function of the endothelial layer might be as important as the risk of interstitial fibrosis. As these effects were not detected for PGE1 this drug should be preferred to papaverine in clinical practice.

Objectives: Primary cell cultures derived from the corpus cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human penis. Methods: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. Results: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0±0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. Conclusion: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.

Preservation of allograft valves is the most important determinant of their durability. Unprocessed, homovital valve allografts stored at 4 degrees C in nutrient medium have provided superior mid-term results over routinely used cryopreserved or antibiotic-sterilized allografts. As storage temperature may alter viability, it was hypothesized that allograft storage at 37 degrees C may maintain greater viability over time.Porcine aortic (n = 10) and pulmonary valve conduits (n = 10) were harvested under sterile conditions. Valve leaflets and sinus walls were separated, and each was divided into two specimens, which were stored in modified culture medium at 4 degrees C and 37 degrees C, respectively. Cell viability was tested by monitoring metabolic activity at 37 degrees C at days 1, 3, 7, 10, and 14. The proliferative ability of cells isolated from valve leaflets was assessed after 14 days by cell culture. Sterility testing of the storage medium was also carried out.Valve leaflet cells and sinus wall cells had significantly higher metabolic activity when stored at 37 degrees C. The median number of isolated cells at 4 degrees C was 3,231.5 (range: 422-3,844), and at 37 degrees C was 8,317.50 (range: 4,329-8,650). The storage medium was sterile in all cases.Storage at 37 degrees C significantly improved valve allograft cell metabolic activity and viability compared with storage at 4 degrees C for up to 14 days. The lower concentration of antibiotics did not affect the sterility of tissues stored at 37 degrees C.

Lymphangiomatosis is a rare disease characterised by multiple, infiltrative proliferations of lymphatic endothelium in the mediastinum and other somatic or visceral sites. Surgical resection, chemotherapy, and radiotherapy are all used to treat this disorder but have limited effectiveness. We have characterised the morphological and functional endothelial properties of the lymphatic tissue of one patient with mediastinal lymphangiomatosis and identified two morphologically and functionally different types of lymphatic endothelial cell (LECI and LECII). The morphology and cellular function of LECI cells resembled vascular endothelial cells and lymphatic tissues of healthy individuals (figure A; stained with an antibody against epidermal growth-factor [EGF] receptor). LECII cells, however, had increased proliferation and an altered immunohistochemical appearance—in particular, pronounced expression of CD31 and EGF-receptor (figure B; stained with an antibody against EGF-receptor). Increased expression of CD31 in LECII cells suggests they can be targeted with non-specific antiangiogenetic agents such as interferons or vascular endothelial growth-factor inhibitors. Furthermore, increased expression of EGF-receptors in LECII cells could provide a therapeutic target for selective EGF-receptor tyrosine-kinase inhibitors. These data show that mediastinal lymphangiomatosis is associated with an aberrant lymphatic endothelial cell-type that has targetable characteristics allowing for disease-specific pharmacological intervention.

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.

Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1 ), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the corpus cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the corpus cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1 , 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1 . Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the corpus cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.

Disadvantages associated with commercially available vascular implants necessitate alternative strategies to develop new vascular prostheses. Although many tissue characterizing strategies have been defined, no valid test for thrombogenicity exists. Here we introduce a novel concept for thrombogenicity testing of vascular implantsSilastic tubes were implanted into the carotid arteries of 12 sheep. After placing these shunts, tc99m-labeled platelets were administered and test-vessels were put in between the shunts. Native autologous (n=6), as well as native/acellularized allogeneic (n=6/n=6), and xenogeneic (n=6/n=6) carotid arteries and allogeneic (n=6/n=6) and xenogeneic (n=6/n=6) carotid arteries reseeded with allogeneic endothelial-cells, fibroblasts and myocytes were evaluated. Number and time course of intra-operatively deposited platelets were evaluated with a Geiger-counter; certain areas of platelet deposition located, envisioned and characterized by a gamma-camera and scanning electron-microscopy afterwards.Counter results revealed no significant different platelet depositions when comparing silastic tubes with either autologous or allogeneic native carotid arteries. However, starting 5 minutes after placement, acellularized/reseeded allogeneic (p=0.001/p=0.00004), and xenogeneic (p=0.0001/p=0.01) carotid arteries showed significantly more platelet depositions than native autologous carotides. Moreover, it was possible to show that almost no platelets adhere to native vessels or silastic tubes, thus proving the test method itself.The Ex-Vivo-Shunt-Model is a valid method to measure and envision the intrinsic thrombogenicity of vascular implants.

Objectives: The aim of the present study was to generate a tissue engineered type of mechanically stable graft suitable for surgical replacement of the tunica albuginea penis. Methods: Porcine fibroblasts isolated from open fascia biopsies were seeded on decellularized collagen matrices and then cultivated in a bioreactor under continuous multiaxial stress for up to 21 days ( n =12). Static cultures without mechanical stress served as controls. Cell proliferation, cell alignment, and de novo synthesis of extracellular matrix proteins (proteoglycans, procollagen I, elastin) in these grafts was evaluated by hematoxylin-eosin, pentachrome, and immuno-staining. Additionally, the enzymatic isolation of porcine fibroblasts from O4 mm skin punch biopsies ( n =8) was evaluated. Results: Mechanically strained cultures of fibroblasts showed a homogeneous multilayer matrix infiltration and a regular cell alignment in the direction of strain axis after 7 days, as well as a de novo production of extracellular matrix proteins compared to the static control. A large amount of viable fibroblasts was easily obtained from small skin punch biopsies. Conclusion: This study shows that continuous multiaxial stimuli improve proliferation and extracellular matrix synthesis of mature fibroblasts reseeded on a biological matrix making this a feasible autologous tissue engineered graft for penile surgery. For the clinical setting fibroblasts harvested from small skin biopsies can be a comfortable cell source.

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.

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.

Objective: Acellularised porcine scaffolds have been successfully used for cardiovascular tissue engineering. However, there is concern about the possibility of porcine endogenous retrovirus (PERV) transmission. In this study we developed an in vivo model for cross-species PERV transmission. Methods: In vitro autologous repopulated porcine pulmonary arteries ðn ¼ 6Þ were implanted in sheep in orthotopic position. Blood samples were collected regularly up to 6 months after implantation and tested for PERV by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. Explanted tissue engineered pulmonary arteries were tested for PERV sequences. Results: PERV DNA was detectable in acellularised porcine scaffolds. No PERV sequences were detectable 6 months after implantation of in vitro repopulated acellularised porcine pulmonary arteries and in all tested peripheral blood samples. Conclusions: Acellularised porcine matrix scaffolds can be used for cardiovascular tissue engineering of autologous grafts without risk of PERV transmission.

Despite the central and peripheral effects of androgens on the nervous system, the local effects of androgens in the corpus cavernosum penis and their importance for erectile function is still unclear. In this study corpus cavernosum biopsies of eight adult potent patients, aged 19-63 years, undergoing penile deviation surgery (group A) and 12 patients undergoing male-to-female transsexual surgery (group B) were immunostained for nuclear androgen and estrogen-alpha receptors. Additionally, primary corpus cavernosum endothelial cell cultures were obtained from six transsexual patients and exposed to testosterone, dihydrotestosterone, estradiol and progesterone likewise for 7 days. Total cell count was performed and cell metabolic activity was measured by a tetrazolium salt-based assay. Androgen and estrogen-alpha receptors were detected in stromal as well as in endothelial cells. Of all cell nuclei, 74.9% (SD 16.4) in group A and 63.5% (SD 17.1) in group B were positively stained for androgen receptors. The respective percentage of estrogen receptors was 11% (SD 9.5) and 21.2% (SD 12.6). An age-dependent difference in receptor distribution was not observed in either group. In the cell culture system only cultures exposed to testosterone and dihydrotestosterone showed a dose-dependent increase of cell metabolic activity compared to the cultures supplemented with estradiol and progesterone. The significant and age-independent high androgen and low estrogen-alpha receptor distribution found in both groups suggests a possible peripheral effect of androgens at the level of the corpus cavernosum penis in adult humans. This is supported by the observed effect of testosterone and dihydrotestosterone on cell count and endothelial cell metabolism in our cell culture system. The role of estrogens remains unclear.

Biological heart valve prostheses undergo degenerative changes which lead ultimately to dysfunction or even complete destruction. The study aim was to evaluate immunological factors and their potential role in biological heart valve destruction.Allogeneic (n = 10) and xenogeneic (n = 3) aortic valve prostheses, as well as aortic valves retrieved from transplanted human hearts which had to be replaced due to chronic graft rejection (n = 4), were analyzed. Aortic valves from human donor hearts (n = 4) served as controls. Evaluated adhesion molecule expression included: selectin family ELAM-1, CD62; integrin family VLA-1, -2, -3, -4, -5 and -6; immuoglobulin supergene family PECAM-1, ICAM-1 and -2, and class I heavy chain proteins; complementary adhesion molecules CD34, CD44 and von Willebrand factor.ELAM-1, ICAM-1 and -2, CD34, CD44 and class I heavy chain proteins, which play significant roles during inflammatory processes, showed stronger expression patterns in allogeneic and xenogeneic aortic heart valve prostheses compared to native or chronically rejected valves. Furthermore, allogeneic and xenogeneic valves showed a strong thrombogenicity which stained positive for von Willebrand factor outside endothelial cells on these valves. Integrin molecules as well as CD62 showed only mild differences.Immunological reactions play a pivotal role in the degeneration of biological heart valve prostheses. As immunosuppressive therapy after heart valve replacement is not a viable option, novel approaches in 'tissue engineering' may help to avoid tissue degeneration while preserving the advantage of biological tissue origin.

Objective Commercially available biological heart valve prostheses undergo degenerative changes, which finally lead to complete destruction. Here we evaluate the role of inflammation and ischemia after implantation of xenogeneic heart valve conduits (XPC) generated by novel concepts of tissue engineering. Methods Acellularized (a-)XPC and autologus re-seeded (s-)XPC were implanted into sheep. Samples were taken as follows: after acellularization (n=2), after re-seeding (n=2), 6 months (seeded/non-seeded: n=3/5), 9 months (n=2/5), and 12 months (n=3/2) post implantation. Five native porcine conduits served as control. Using histological methods, samples were evaluated for pathological changes and existence/density of microvessels. Results Prior to implantation a-XPC were completely free of cells. Six months after implantation, leaflets and pulmonary arteries of s-XPC and a-XPC showed good endothelial surface coverage. Microvessel density within the myocardial cuffs and pulmonary vessel walls were comparable to control in all grafts. However, 6, 9 and 12 months after implantation pathological severe microvessel ingrowth, calcification and cellular infiltrations were observed on a-XPC and s-XPC valves, whereas myocardial cuffs and XPC-artery walls showed only mild degenerative alterations. Conclusions Inflammatory reactions play a pivotal role in the degeneration of a-XPC and s-XPC. Thus, since ischemia seems to have little or no influence on this process, inflammation inductive factors should be the center of interest.

Objective: to develop a graft bearing an immunologically tolerated tissue-engineered venous valve (TE graft) that will be incorporated into a native vessel, and restore normal valve function for the treatment of chronic venous insufficiency. Methods: twenty-four TE grafts were grown using decellularised allogeneic ovine veins as donor matrix, which was subsequently repopulated with the future recipient's myofibroblasts (MFB) and endothelial cells (EC). TE grafts were implanted into the external jugular vein. Animals were sacrificed at 1, 6, and 12 weeks ( n = 4, each). Autografts served as controls (1 week, n = 4; 6 weeks, n = 4). Specimen for histology and immunohistochemistry were taken. Results: the matrix was successfully repopulated with MFB and EC ( n = 8). Patency on venography in the TE graft-group was 4/4, 4/4, and¾ at 1, 6, and 12 weeks, and 4/4 (4/4) in autografts at 1 (6) weeks, respectively. Except for 2 TE grafts after 12 weeks, valves were competent (duplex ultrasound). Patent TE grafts were merely distinguishable from autografts with minor inflammatory reactions. Reflux was caused by neo-intima formation related to the basis of the TE graft. Conclusion: acellularisation and consecutive in vitro autogeneic re-seeding of valved venous conduits can lead to immunologically acceptable, patent, and competent implants in sheep. Eur J Vasc Endovasc Surg 25 , 305-312 (2003)

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: 3.0–3.8 mm; CA: 0.7–0.88 mm; JV: 0.35–0.61 mm). 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.

Regenerative medicine and reconstructive surgery is often limited by the availability of normal tissue. Tissue engineering provides promise in the development of “artificial tissues”. Recent advances in the field of tissue engineering have made the goal of constructing whole organs and tissue types to replace damaged, injured or missing body tissue with biologically compatible substitutes. To create neo-organs two components are essential. Scaffolds to carry the second component the cells. To ensure survival of the bioartifical tissue the scaffold matrix components must be remodeled. Within this process matrix metalloproteinases (MMPs) are the key enzymes. Naturally matrix metalloproteinases are the major proteins in the cell associated extracellular-matrix-remodeling of physiological and pathological processes. They share a highly conserved structure of the individual domains with a number of identifiable modules linked to their specific functions.

Objective Tissue engineered heart valves based on polymeric or xenogeneic matrices have several disadvantages, such as instability of biodegradable polymeric scaffolds, unknown transfer of animal related infectious diseases, and xenogeneic rejection patterns. To overcome these limitations we developed tissue engineered heart valves based on human matrices reseeded with autologous cells. Methods and Results Aortic (n=5) and pulmonary (n=6) human allografts were harvested from cadavers (6.2±3.1 hours after death) under sterile conditions. Homografts stored in Earle’s Medium 199 enriched with 100 IU/mL Penicillin-Streptomycin for 2 to 28 days (mean 7.3±10.2 days) showed partially preserved cellular viability (MTT assay) and morphological integrity of the extracellular matrix (H-E staining). For decellularization, valves were treated with Trypsin/EDTA resulting in cell-free scaffolds (DNA-assay) with preserved extracellular matrix (confocal microscopy). Primary human venous endothelial cells (HEC) were cultivated and labeled with carboxy-fluorescein diacetate-succinimidyl ester in vitro. After recellularization under fluid conditions, EC were detected on the luminal surfaces of the matrix. They appeared as a monolayer of positively labeled cells for PECAM-1, VE-cadherin and Flk-1. Reseeded EC on the acellular allograft scaffold exhibited high metabolic activity (MTT assay). Conclusions Earle’s Medium 199 enriched with low concentration of antibiotics represents an excellent medium for long time preservation of extracellular matrix. After complete acellularization with Trypsin/EDTA, recellularization under shear stress conditions of the allogeneic scaffold results in the formation of a viable confluent HEC monolayer. These results represent a promising step toward the construction of autologous heart valves based on acellular human allograft matrix.