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Dr. Johannes Wilken


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Veröffentlichungen aus 2008 / Fakultät IA

Experimental analysis of biaxial mechanical tension in cell monolayers and cultured three-dimensional tissues: The CellDrum Technology / von Jürgen Trzewik
Ilmenau : Univ.-Verl. Ilmenau, 2008. - IX, 126 S.
ISBN 978-3-939473-26-8
Preis: 18,60 €

Zugl.: Ilmenau, Techn. Univ., Diss., 2007

A person’s health is determined by the biomechanical integrity and performance of tissue and organs. Widespread diseases like hernia formation or pelvic floor malfunction are directly related to a reduced biomechanical functionality of the concerned soft tissue. These exemplary diseases, but also dysfunction of the cardiovascular system are caused by processes, which are based on changes of biomechanical properties at the cellular level. Fundamental research in this topic on humans is limited for practical and ethical reasons, especially the evaluation of active substances. The systems for the in vitro evaluation of biomechanical material properties, as described in the literature, are adopted from standard material test methods mainly focusing on an uniaxial loading. But uniaxial loading is rarely seen in nature since most load cases are two or three dimensional. This work describes a new measurement principle, which allows the cultivation of cell monolayers or thin tissue composites under biaxial load conditions and mechanical evaluation at the same time. A new cell cultivation module, termed CellDrum, was developed for that purpose. The Celldrum consists of a thin, biocompatible silicon membrane attached to a cylindrical well . The CellDrum membrane can be populated with cells grown in monolayer structures or serves as a sealing boundary layer for thin film cell-matrix constructs anchored with the Cell Drum’s wall. The CellDrum technology is a universal approach and can be used for almost any kind of material test on thin films. Investigations can be performed either under a dynamic or quasi-statical mode. Mechanical properties of cell populated CellDrum constructs were analyzed in the dynamic setup, after excitation via a pressure impulse and measuring the resonance frequency of vibrating tissue constructs anchored to the CellDrum. The quasi-statical mode allows evaluating the stress-strain behavior of test material (in this work: cell monolayer and thin tissue equivalents). Technologies were developed, adapted to the specific questions and experimental needs, ranging from flexible, µm-thin, biocompatible membranes over hard- and software solutions up to the biomechanical analyses for the extraction of adequate biomechanical parameters. This interdisciplinary effort between cell- and molecular biology, polymer chemistry, mechanics and other engineering fields, which had to be controlled in equal measure, was the major challenge of this work.Optical monitoring during cell cultivation is vitally important for the development, standardization and optimization of cell-biological measurement protocols. The possibility of a continuous microscopic evaluation of the test specimen, at a minimal disturbance of the measurement environment, is a unique feature of the described apparatus. In combination with confocal laser microscopy it is also possible to identify cell density and orientation during the cultivation period. These values are important parameters for the correct interpretation of the derived measurement data.Cell based systems can sense changes in their mechanical environment and promote alterations and adaptations in tissue structure and function, like metabolism, orientation, protein and gene expression. These factors were considered for the CellDrum development and carefully evaluated by accompanying experiments. The use of circular CellDrum constructs introduces a homogenous stress distribution within the sample construct, similar to the biaxial state found in most in vivo situations for soft tissues. The CellDrum system therefore supports a cell proliferation with random cell orientation in contrast to the initiation of parallel cell orientation found in common approaches with rectangular stretched cell seeded collagen cells. Furthermore, finite element method investigations proved the homogenous stress distribution for the circular CellDrum, compared to the strong variations of the van Mieses equivalent stress values in a rectangular stretched gel. Besides mentioned circumstances, further experiments had to be performed to evaluate the contributions of different membrane coatings on the cellular response to substrate strain variations. The cells reacted differently to the employed coating under strained and unstrained conditions. The measurement system described in this work offers the opportunity to analyze the mechanical properties of cell constructs over a long period of time (weeks) under defined mechanical boundary conditions either in the dynamic or statistical setup. The relation between cell proliferation and residual tissue stress was exemplarily demonstrated. The studied cellular response, especially the plateau effect during the generation of tissue tension, provides further conclusions about mechanical homeostasis of soft tissue. The understanding of these phenomena may contribute to the understanding of the mechanics of wound healing and of scar formation. The stimulation of tissue equivalents during cell cultivation and the variation of process parameters had a direct impact on the E-module and residual tension of the evaluated specimen. Both parameters were analyzed synchronously by the transfer and adequate adaptation of an analyzing method, which was originally developed for microsytem technologies. Furthermore, described system is the first system which offers the novel and unique possibility to cultivate synchronously beating cardiomyocytes and evaluate the contractile behaviour together with the monitored beating frequency. A specific method was developed to obtain primary cardiomyocytes from 10 day old chicken embryos and later on transferred by a research partner on neonatal rat myocytes. Complicated and error-prone experimental handling was a major concern in earlier methods, besides the unsatisfying situation of undefined mechanical boundary conditions. During pharmaceutical research activities, thousands of substances have to be evaluated for potential pharmaceutically efficacy by secondary screening. Unlike most devices introduced before, the system established here can easily be scaled up towards a high-throughput system providing simultaneous material data on large numbers of tissue constructs exposed to various chemical agents. This was exemplarily demonstrated for fibroblast-collagen composites.

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