K Gamerdinger

Characteristics of highly flexible PDMS membranes for long-term mechanostimulation of biological tissue.

Measurement of mechanical properties of soft biological tissue remains a challenging task in mechanobiology. Recently, we presented a bioreactor for simultaneous mechanostimulation and analysis of the mechanical properties of soft biological tissue samples. In this bioreactor, the sample is stretched via deflection of a flexible membrane. It was found that the use of highly compliant membranes increases accuracy of measurements. Here, we describe the production process and characteristics of thin and flexible membranes of polydimethylsiloxane (PDMS) designed to improve the signal-to-noise ratio of our bioreactor. By a spin-coating process, PDMS membranes were built by polymerization of a two component elastomer. The influence of resin components proportion, rotation duration, and speed of the spinning were related to the membrane mechanics. Membranes of 22 mm inner diameter and 33 to 36 microm thickness at homogeneous profiles were produced. Isolated rat diaphragms served as biological tissue samples. Mechanical properties of the membranes remained constant during 24 h of mechanostimulation. In contrast, time- and strain-dependent mechanical properties of the diaphragms were found.

Mechanostimulation, electrostimulation and force measurement in an in vitro model of the isolated rat diaphragm

In an in vitro model of the entire rat diaphragm, diaphragmatic contraction forces at defined preload levels were investigated. A total of 24 excised rat diaphragms were electrically stimulated inside a two-chamber strain-applicator. The resulting contraction forces were determined on eight adjusted preload levels via measuring the elicited pressure in the chamber below the diaphragm. Subsequently, diaphragms were exposed for 6 h to one of four treatments: (1) control, (2) cyclic mechanical stretch, (3) intermittent electrical stimulation or (4) combination of cyclic mechanical stretch and electrical stimulation. Diaphragmatic contraction force increased from 116 ± 21 mN at the lowest preload level to 775 ± 85 mN at the maximal preload level. After 6 h maximal muscle contraction forces were smallest after non-electrostimulated treatment (control: 81 ± 15 mN, mechanical deflection: 94 ± 12 mN) and largest after electrostimulation treatment (mere electrostimulation: 165 ± 20 mN, combined mechano- and electro-stimulation: 164 ± 14 mN). We conclude that our model allows force measurements on isolated rat diaphragms. Furthermore, we conclude that by intermediate electrical stimulation diaphragmatic force generation was better preserved than by mechanical stimulation.

Mechanical load and mechanical integrity of lung cells – Experimental mechanostimulation of epithelial cell- and fibroblast-monolayers

Experimental mechanostimulation of soft biologic tissue is widely used to investigate cellular responses to mechanical stress or strain. Reactions on mechanostimulation are investigated in terms of morphological changes, inflammatory responses and apoptosis/necrosis induction on a cellular level. In this context, the analysis of the mechanical characteristics of cell-layers might allow to indicate patho-physiological changes in the cell-cell contacts. Recently, we described a device for experimental mechanostimulation that allows simultaneous measurement of the mechanical characteristics of cell-monolayers. Here, we investigated how cultivated lung epithelial cell- and fibroblast-monolayers behave mechanically under different amplitudes of biaxial distension. The cell monolayers were sinusoidally deflected to 5%, 10% or 20% surface gain and their mechanical properties during mechanostimulation were analyzed. With increasing stimulation amplitudes more pronounced reductions of cell junctions were observed. These findings were accompanied by a substantial loss of monolayer rigidity. Pulmonary fibroblast monolayers were initially stiffer but were stronger effected by the mechanostimulation compared to epithelial cell-monolayers. We conclude that, according to their biomechanical function within the pulmonary tissue, epithelial cells and fibroblasts differ with respect to their mechanical characteristics and tolerance of mechanical load.

The Equilibration of PCO2 in Pigs Is Independent of Lung Injury and Hemodynamics.

Objectives: In mechanical ventilation, normoventilation in terms of PCO2 can be achieved by titration of the respiratory rate and/or tidal volume. Although a linear relationship has been found between changes in respiratory rate and resulting changes in end-tidal cO2 (△PetCO2) as well as between changes in respiratory rate and equilibration time (t eq) for mechanically ventilated patients without lung injury, it is unclear whether a similar relationship holds for acute lung injury or altered hemodynamics. Design: We performed a prospective randomized controlled animal study of the change in PetCO2 with changes in respiratory rate in a lung-healthy, lung-injury, lung-healthy + altered hemodynamics, and lung-injury + altered hemodynamics pig model. Setting: University research laboratory. Subjects: Twenty mechanically ventilated pigs. Interventions: Moderate lung injury was induced by injection of oleic acid in 10 randomly assigned pigs, and after the first round of measurements, cardiac output was increased by approximately 30% by constant administration of noradrenalin in both groups. Measurements and Main Results: We systematically increased and decreased changes in respiratory rate according to a set protocol: +2, -4, +6, -8, +10, -12, +14 breaths/min and awaited equilibration of Petco2. We found a linear relationship between changes in respiratory rate and △PetCO2 as well as between changes in respiratory rate and t eq. A two-sample t test resulted in no significant differences between the lung injury and healthy control group before or after hemodynamic intervention. Furthermore, exponential extrapolation allowed prediction of the new PetCO2 equilibrium and t eq after 5.7 ± 5.6 min. Conclusions: The transition between PetCO2 equilibria after changes in respiratory rate might not be dependent on moderate lung injury or cardiac output but on the metabolic production or capacity of cO2 stores. Linear relationships previously found for lung-healthy patients and early prediction of PetCO2 equilibration could therefore also be used for the titration of respiratory rate on the PetCO2 for a wider range of pathologies by the physician or an automated ventilation system.

Mechanical properties of human lung cells after mechanostimulation

Experimental mechanostimulation of soft biologic tissue is a widely used method to analyze cellular reactions to stress or strain. While there are plenty different methods to stimulate cells or tissues and thereby to analyze intracellular and extracellular processes, it remains difficult to analyze the forces produced by cells or tissues while cells resist the applied strain. We previously described a mechanostimulator [1] for strain application to soft biologic samples. Here we show how lung epithelial cells that are cultivated on self-made highly flexible RGD-peptide surface treated PDMS membranes behave under increasing surface-deflection. We reveal that a decrease in stiffness (increase in compliance) of pulmonary epithelial cell layers after stimulation comes along with a change in cell adherence and loss of cell-cell junc-

Ventilation-analogue mechanostimulation of lung epithelial cells in vitro

During spontaneous breathing the mechanical strain on the pulmonary tissue is akin to a sinusoidal profile. In contrast, during mechanical ventilation the stimulation profile of the lung tissue differs considerably from a sinusoidal pattern as it can be shown analyzing the frequency contents of the ventilatory pattern by means of a Fourier-Transformation. In vitro used methods for mechanostimulation of soft biologic tissue for the analysis of cellular reactions to dynamic stress or strain use exclusively sinusoidal stimulation patterns. In this work for the first time we compare reactions of lung epithelial cells in vitro stimulated either with sinusoidal or ventilation-analogue mechanostimulation profile. Our previously published mechanostimulator-system allows not only for stimulating the cells but thereby also for analyzing the mechanical properties of cell layers.

Mechanostimulation and Mechanics Analysis of Lung Cells, Lung Tissue and the Entire Lung Organ

Analysis of respiratory mechanics under mechanical ventilation is crucial for a lung-protective ventilation setting. However, under the conditions of mechanostimulation caused by mechanical ventilation, only the global components of mechanical impedance can be determined. These include the airflow resistance, compliance, and inertance. Whereas in the case of conventionalmechanical ventilation, the organ integrity of the lung is certainly preserved, it is practically impossible to obtain quantitative information about the local pulmonary mechanics, for instance at the alveolar level. Analysis of pulmonary mechanics at a local level requires sophisticated experimental techniques for the mechanostimulation of anatomical subunits of the lung. In this chapter, we summarize our investigations in the field of experimental mechanostimulation and mechanics analysis of lung cells, lung tissue and entire lung organ.