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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.
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Prótese Vascular , Artérias Carótidas/citologia , Tecido Conjuntivo/química , Células Endoteliais/citologia , Músculo Liso Vascular/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Artérias Carótidas/ultraestrutura , Linhagem Celular , Elasticidade , Células Endoteliais/transplante , Humanos , Músculo Liso Vascular/transplante , Suínos , Resistência à TraçãoRESUMO
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 < 5 mm 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 α-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.
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Artérias , Prótese Vascular , Células Endoteliais , Músculo Liso Vascular , Miócitos de Músculo Liso , Engenharia Tecidual/métodos , Animais , Movimento Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , SuínosRESUMO
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.
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Aorta/patologia , Valva Aórtica/patologia , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Ácido Edético/química , Células Endoteliais/citologia , Fibroblastos/metabolismo , Masculino , Modelos Animais , Células Musculares/citologia , Ratos , Ratos Endogâmicos Lew , Alicerces Teciduais/química , Tripsina/químicaRESUMO
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.
Assuntos
Retrovirus Endógenos/fisiologia , Fígado Artificial/virologia , Suínos/virologia , Animais , Células Cultivadas , Células Endoteliais/virologia , Hepatócitos/virologia , HumanosRESUMO
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.
Assuntos
Órgãos Bioartificiais , Engenharia Tecidual/métodos , Transplante Autólogo/métodos , Animais , Endotélio Vascular/transplante , Matriz Extracelular/transplante , Humanos , Jejuno/irrigação sanguínea , Jejuno/transplante , Ratos , SuínosRESUMO
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.
RESUMO
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 harboring 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.
Assuntos
Capilares/citologia , Matriz Extracelular/fisiologia , Hepatócitos/metabolismo , Fígado Artificial , Fígado/citologia , Pesquisa , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Géis , Imuno-Histoquímica , Jejuno/irrigação sanguínea , Lactatos , Modelos Biológicos , Sus scrofa , Fatores de Tempo , Ureia/metabolismoRESUMO
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 tissuespecific 3D test systems may accelerate the progress in design and development of cancer therapies.
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Antineoplásicos/farmacologia , Órgãos Bioartificiais , Neoplasias/tratamento farmacológico , Engenharia Tecidual , Pesquisa Biomédica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Proteômica , Ensaio Tumoral de Célula-TroncoRESUMO
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 m2 and 1.07 to 1.46 m2, 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.
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Bioprótese , Implante de Prótese de Valva Cardíaca , Próteses Valvulares Cardíacas , Monócitos/transplante , Insuficiência da Valva Pulmonar/cirurgia , Valva Pulmonar/cirurgia , Engenharia Tecidual , Adolescente , Reatores Biológicos , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/transplante , Criança , Estudos de Viabilidade , Feminino , Seguimentos , Humanos , Masculino , Monócitos/citologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Artéria Pulmonar/anormalidades , Valva Pulmonar/anormalidades , Insuficiência da Valva Pulmonar/etiologia , Tetralogia de Fallot/cirurgia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Transplante Autólogo , Transplante Homólogo , Resultado do TratamentoRESUMO
BACKGROUND: Tissue-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. METHODS: Ovine 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. RESULTS: The 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. CONCLUSIONS: Tissue-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.
Assuntos
Cardiopatias Congênitas/cirurgia , Artéria Pulmonar , Engenharia Tecidual , Animais , Ovinos , Engenharia Tecidual/métodosAssuntos
Órgãos Artificiais , Músculo Esquelético/fisiologia , Músculo Esquelético/transplante , Transplante Autólogo/métodos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Divisão Celular , Empiema Pleural/etiologia , Empiema Pleural/cirurgia , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos/métodos , RadiografiaRESUMO
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.
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Engenharia Tecidual/métodos , Animais , Antígenos CD , Órgãos Bioartificiais , Células da Medula Óssea/citologia , Caderinas/química , Adesão Celular , Diferenciação Celular , DNA/química , Endotélio/citologia , Endotélio Vascular/metabolismo , Fluordesoxiglucose F18/química , Imuno-Histoquímica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Tomografia por Emissão de Pósitrons , Regeneração , Células-Tronco/citologia , Suínos , Fatores de TempoRESUMO
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.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Pênis/patologia , Alprostadil/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/fisiologia , Imunofluorescência , Humanos , Técnicas In Vitro , Masculino , Papaverina/farmacologia , Fentolamina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Vasodilatadores/farmacologiaRESUMO
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.
Assuntos
Endotélio Vascular/citologia , Pênis/citologia , Células Estromais/citologia , Engenharia Tecidual , Adulto , Idoso , Pesquisa Biomédica , Western Blotting , Técnicas de Cultura de Células/métodos , Células Cultivadas , Endotélio Vascular/efeitos da radiação , Disfunção Erétil/patologia , Disfunção Erétil/terapia , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Humanos , Imuno-Histoquímica , Magnetismo , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Músculo Liso/citologia , Músculo Liso/efeitos da radiação , Reprodutibilidade dos Testes , Células Estromais/efeitos da radiaçãoRESUMO
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.
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Órgãos Bioartificiais , Engenharia Tecidual , Bexiga Urinária , Animais , Músculo Liso/citologia , Urotélio/citologia , Grau de Desobstrução VascularRESUMO
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.
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Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cocultura/instrumentação , Miócitos Cardíacos/citologia , Engenharia Tecidual/instrumentação , Animais , Animais Recém-Nascidos , Dióxido de Carbono/análise , Contagem de Células , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Meios de Cultura , Meios de Cultivo Condicionados/química , Metabolismo Energético , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Miócitos Cardíacos/metabolismo , Oxigênio/análise , Pressão Parcial , Fluxo Pulsátil , Ratos , Ratos WistarRESUMO
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.