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Heike Mertsching

Publications and source records attributed to Heike Mertsching.

At least 19 recordsLinked to original sources
PubMed2006

Clinical application of tissue engineered human heart valves using autologous progenitor cells.

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.

Adolescent↗
PubMed2006

Tissue engineering of viable pulmonary arteries for surgical correction of congenital heart defects.

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.

Animals↗
PubMed2005

A multifunctional bioreactor for three-dimensional cell (co)-culture.

Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown. Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4',6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14. The optimized seeding density in the matrix was 2.0 x 10(7) cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2-183.7 +/- 18.4 mmHg) and physiological pH values (7.4 +/- 0.02) and a constant level of pCO2 (43.1 +/- 2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82 +/- 6.7% living cells. No significant differences were found between spatial separated bioreactor compartments. Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.

Animals↗
PubMed2005

Engineering of a vascularized scaffold for artificial tissue and organ generation.

Tissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However, complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized by quantitative 2-[18F]-fluoro-2'-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistological work up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascular structures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding. Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate that our generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinical application.

Animals↗
PubMed2005

Isolation of primary endothelial and stromal cell cultures of the corpus cavernosum penis for basic research and tissue engineering.

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.

Adult↗
PubMed2005

Biological vascularized matrix for bladder tissue engineering: matrix preparation, reseeding technique and short-term implantation in a porcine model.

PURPOSE: We generated a vascularized, autologous, reseeded bladder substitute and evaluated immediate vascularization and perfusion of the graft after implantation to the recipient organism in a porcine model. MATERIAL AND METHODS: Acellular matrix was processed from porcine small bowel segments by subsequent mechanical, chemical and enzymatic decellularization, preserving the jejunal arteriovenous pedicles. In 2 separate steps the matrix was reseeded with primary bladder smooth muscle cells (SMCs) and urothelial cells (UCs), and its vascular structures were resurfaced with endothelial progenitor cells (EPCs). To evaluate graft perfusion short-term implantation was performed. RESULTS: The acellular scaffold was successfully repopulated with multilayers of ingrowing SMCs and superficial UCs. After reseeding the jejunal arteriovenous pedicles with EPCs and cultivation for 3 weeks the larger vessels as well as the intramural scaffold capillary network were repopulated with cell monolayers expressing endothelial specific proteins. Perfusion stagnation and implant thrombosis occurred within 30 minutes after the implantation of acellular scaffolds not reseeded with EPCs. In the EPC reseeded group the vascular system revealed intact perfusion and no relevant thrombus formation was observed after 1 or 3 hours. CONCLUSIONS: The current study of successful SMC and UC reseeding, vessel resurfacing with EPCs and short-term vascular patency represents the promising in vitro and in vivo basis for further evaluation of this biological vascularized matrix in chronic long-term large animal implantation experiments.

Animals↗
PubMed2005

In vitro viability of human cavernosal endothelial and fibroblastic cells after exposure to papaverine/phentolamine and prostaglandin E1.

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.

Alprostadil↗
PubMed2004

Functional neointima characterization of vascular prostheses in human.

BACKGROUND: The purpose of this study was to evaluate neointimal functionality of synthetic vascular grafts repopulated by host cells after implantation. METHODS: We obtained reseeded inflow and outflow cannulas of 2 patients undergoing orthotopic heart transplantation after left ventricular assist device implantation 9 and 10 months before. After cell isolation we examined cellular function of reseeded cells and their capability to form a functional endothelial layer applying immunohistologic markers and quantitative Western blot for endothelial nitric oxide synthase activity. RESULTS: Neointima formation in inflow and outflow cannulas differs macroscopically and by histologic appearance. The neointima formation on the surface of the polyethylene terephthalate fiber (Dacron) grafts differs substantially from native aortic vessel wall with respect to cellular and extracellular matrix composition and cellular function. CONCLUSIONS: The neointima of Dacron prostheses is composed of cells with rudimentary physiologic endothelial function. We conclude that synthetic matrices are not suitable scaffolds for generating functional cardiovascular implants.

Blood Vessel Prosthesis↗
PubMed2004

Expansion of chondrocytes in a three-dimensional matrix for tracheal tissue engineering.

BACKGROUND: The generation of autologous tracheal implants by tissue-engineering techniques is a promising concept for otherwise untreatable patients. A functional cartilaginous backbone represents a prerequisite for any bioartificial tracheal graft. The aim of this study was to define suitable cell types and culture conditions for the generation of tracheal cartilage. METHODS: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot. RESULTS: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis. CONCLUSIONS: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering.

Animals↗
PubMed2004

Guided tissue regeneration: porcine matrix does not transmit PERV.

OBJECTIVE: For cardiovascular tissue engineering, acellularized scaffolds of porcine matrices have been successfully used. However, the possibility of porcine endogenous retrovirus (PERV) transmission remains debatable. In this study, we investigated whether acellularized porcine vascular scaffolds cause cross-species transmission of PERV in a xenogenic model. METHODS: Porcine pulmonary arteries were acellularized and implanted into sheep in orthotopic position (n=6). Cardiopulmonary bypass support was used for all operations. Blood samples were collected regularly up to 6 months after the operation, and cellular components were tested for PERV infection by PCR and RT-PCR. Grafts were explanted 6 and 12 months after implantation. Tissue samples were characterized by histology and electron microscopy and tested for PERV sequences. RESULTS: All animals survived the procedure and follow up until explantation of the grafts. PERV DNA was detectable in acellularized scaffolds of porcine matrices. Acellular porcine pulmonary arteries scaffolds were repopulated in vivo by autologous cells of the host, leading to a vessel consisting of all cellular components of the vessel wall. No PERV sequences were detectable neither in all tested peripheral blood samples nor in tissue samples of in vivo recellularized grafts up to 6 months after implantation. Electron microscopy revealed no signs of graft infection by retrovirus. CONCLUSIONS: Guided tissue regeneration of acellularized vascular porcine matrix scaffolds leads to structured vessels up to one year without risk of PERV transmisson.

Animals↗
PubMed2004

Functional tissue engineering of autologous tunica albuginea: a possible graft for Peyronie's disease surgery.

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 X4mm 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.

Animals↗
PubMed2004

Experimental generation of a tissue-engineered functional and vascularized trachea.

OBJECTIVE: We sought to grow in vitro functional smooth muscle cells, chondrocytes, and respiratory epithelium on a biologic, directly vascularized matrix as a scaffold for tracheal tissue engineering. METHODS: Ten- to 15-cm-long free jejunal segments with their own vascular pedicle were harvested and acellularized from donor pigs (n = 10) and used as a vascular matrix. Autologous costal chondrocytes, smooth muscle cells, and respiratory epithelium and endothelial progenitor cells were first cultured in vitro and then disseminated on the previously acellularized vascular matrix. Histologic, immunohistologic, molecular imaging, and Western blotting studies were then performed to assess cell viability. RESULTS: The endothelial progenitor cells re-endothelialized the matrix to such an extent that endothelial cell viability was uniformly documented through 2-(18F)-fluoro-2'-deoxyglucose positron emission tomography. This vascularized scaffold was seeded with functional (according to Western blot analysis) smooth muscle cells and successfully reseeded with viable ciliated respiratory epithelium. Chondrocyte growth and production of extracellular cartilaginous matrix was observed as soon as 2 weeks after their culture. CONCLUSIONS: The fundamental elements for a bioartificial trachea were successfully engineered in vitro in a direct vascularized 10- to 15-cm-long bioartificial matrix. Future experimental work will be directed to give them a 3-dimensional aspect and a biomechanical profile of a functioning trachea.

Animals↗
PubMed2004

Cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human corpus cavernosum penis.

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 microg 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.

Alprostadil↗
PubMed2004

Could 37 degrees C storage temperature extend homovital valve allograft viability?

BACKGROUND AND AIM OF THE STUDY: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Animals↗