Biomedical subjects

K Schenke-Layland

Publications and source records attributed to K Schenke-Layland.

6 recordsLinked to original sources

PubMed2005

Multiphoton autofluorescence imaging of intratissue elastic fibers.

Multiphoton induced blue/green autofluorescence by near infrared femtosecond laser pulses has been used to selectively image intratissue elastic fibers in native and tissue engineered (TE) viable heart valves without any invasive tissue removal, embedding, fixation, and staining. Elastic fibers could be clearly distinguished from collagenous structures which emit ultraviolet/violet radiation when excited with intense ultrashort pulses due to second harmonic generation. Deep-tissue three-dimensional imaging of elastic fibers with submicron spatial resolution was performed by optical sectioning of heart valves using a multiphoton laser scanning microscope in connection with a tunable 80 MHz femtosecond laser source. The technology was used to diagnose extracellular matrix structures and cell resettlement of TE heart valves prior implantation. This novel non-invasive method opens the general possibility of high-resolution in situ imaging of elastic fibers, collagen structures and intracellular organelles in living intact tissues without staining.

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PubMed2004

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

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PubMed2004

Tissue engineering of ovine aortic blood vessel substitutes using applied shear stress and enzymatically derived vascular smooth muscle cells.

Compared to native blood vessels, all clinically available blood vessel substitutes perform suboptimally. Numerous approaches to tissue engineer (TE) blood vessels have been pursued using different scaffold materials, cell types, and culture conditions. Several limitations however remain to be overcome prior to the potential application in the arterial system. This study aimed at tissue engineering viable ovine blood vessels suitable for implantation into the systemic circulation of sheep. In recent studies vascular smooth muscle cells (vSMC) were derived by an explant technique. However, in this study we show that homogenous populations of differentiated vSMC were only obtained by enzymatic dispersion as characterized by immunostaining for specific vSMC marker proteins. In contrast the explant method yielded predominantly less differentiated myofibroblast-like cells. Enzymatically derived vSMC were seeded onto P-4-HB scaffolds and incubated either in a pulsatile flow bioreactor or under static conditions. Dynamically cultured TE blood vessel substitutes showed confluent layered tissue formation and were completely water resistant. They displayed significantly increased ECM synthesis, DNA, and protein content as well as vSMC marker expression. Mechanical properties of bioreactor cultured TE blood vessels approached those of native aorta. In conclusion ovine, aortic blood vessel substitutes were successfully created using enzymatically derived vSMC, bioabsorbable scaffolds, and applied shear stress.

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PubMed2003

Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study.

OBJECTIVE: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. METHODS: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin-eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). RESULTS: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. CONCLUSION: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.

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PubMed2003

Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves.

The multidisciplinary research of tissue engineering utilizes biodegradable or decellularized scaffolds with autologous cell seeding. Objective of this study was to investigate the impact of different decellularization protocols on extracellular matrix integrity of xenogeneic tissue by means of multiphoton femtosecond laser scanning microscopy, biochemical and histological analysis. Pulmonary valves were dissected from porcine hearts and placed in a solution of trypsin-EDTA and incubated at 37 degrees C for either 5, 8, or 24 h, followed by a 24 h PBS washing. Native and decellularized valves were processed for histology, DNA, cell proliferation, matrix proteins and biomechanical testing. Multiphoton NIR laser microscopy has been applied for high-resolution 3D imaging of collagen and elastin. Distinct differences in several ECM components following decellularization time were observed. Total GAG contents decreased in a time-dependent manner, with o-sulfated GAGs being more susceptible to degradation than n-sulfated GAGs. Efficiency of insoluble collagen extraction increased proportionally with decellularization time, suggesting ECM-integrity may be compromised with prolonged incubation. Biomechanical testing revealed a gradual weakening of mechanical strength with increased decellularization time. The enzymatic decellularization process of heart valves revealed a time-dependent loss of cells, ECM components and biomechanical strength. In order to avoid any immune response a thorough decellularization of 24 h remains mandatory.

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PubMedDate not supplied

ProteinChip system technology: a powerful tool to analyze expression differences in tissue-engineered blood vessels.

At the time of implantation, tissue-engineered constructs should resemble native tissues as closely as possible. At present, histology and biochemical methods are commonly used to compare tissue-engineered constructs with native tissue. A ProteinChip system based on surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI) has been developed that allows visualization of complex protein profiles from biological samples. The aim of this study was to determine whether the ProteinChip system is a suitable tool with which to compare the protein expression profiles of tissue-engineered aortic blood vessels with native tissues. Tissue-engineered blood vessel substitutes were fabricated with poly-4-hydroxybutyrate scaffolds, ovine vascular cell seeding, and dynamic tissue culture conditions. Engineered, ovine aortic, and carotid tissues were homogenized and total protein was extracted. Samples were analyzed on ProteinChip arrays. Analysis yielded reproducible protein profiles from all samples. About 150 distinct protein peaks were detected. Comparative analysis with ProteinChip software revealed that the protein profiles from native aorta and native carotid arteries were similar whereas early tissue-engineered samples displayed more distinct deviations. In conclusion, ProteinChip system technology is rapid, reproducible, and highly sensitive in highlighting differentially expressed proteins in tissue-engineered blood vessel substitutes.

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