Guiding respiratory epithelium towards directed ciliary function
Lead supervisor: S. Jockenhövel, Co-supervisor: A. Ludwig, Junior supervisor: L. Thiebes Uniklinik RWTH Aachen, Deptartment of Biohybrid & Medical Textiles, Institute of Applied Medical Engineering
Hypothesis: Flow-induced mechanical stimulation is needed for the induction of directed and coordinated ciliary beating.
Background and aim of C3. (A, B) Respiratory epithelium on fibrin gel with supporting cells shows cilia formation in vitro (unpublished). (C)Spiral mucus transportat as an indication of cilia orientation in a well (unpublished). (D) The aim of this project is to define a stimulation procedure for in vitro development of directed ciliary beating for functional tissue engineered constructs.
Background: Ciliary beating is a key feature of the larger airway epithelium. In order to provide mucociliar clearance in vivo, cilia beat synchronously towards the oral cavity to propel mucus out of the respiratory system. When ciliary beating is dysfunctional, mucus accumulates in the airways increasing the risk of pneumonia. Directed and coordinated ciliary beating of the respiratory epithelium is therefore the basis for generating functional tissue-engineered tracheal substitutes. Beating ciliated epithelium was differentiated in vitro on membranes and scaffold materials such as fibrin gel in our lab (Fig. and 87-90). Directed ciliary function, however, has not yet been achieved by us or any other researchers. Cells coordinate within units in close proximity, but a uniform beating in one direction cannot be achieved in multiwell plates. Once fully developed, the basal feet define the beating direction and the cilia do not reorient. Observations during embryogenesis suggest that fluid streams acting as mechanical stimuli induce directed ciliary beating. This coordination by flow was also observed in vitro for ciliated skin cells of amphibian larvae91. Our observations indicate that respiratory eptihelial cell stimulation by shear stresses with air stream in a bioreactor developed in house can lead to directed ciliary beating and, even more importantly, to a directed mucus transport (unpublished). So far, it is not known which kind of stimulation is needed to achieve uniform cilia orientation. Aims: We intend to define an optimized stimulation protocol for in vitro conditioning of respiratory epithelium on bronchotracheal tissue constructs to obtain functional mucociliary competence. We examine how fluid flow and air flow induce directed ciliary beating during epithelial differentiation. In addition, we investigate the influence of defined pathological mechanical stimuli on ciliary function and inflammatory response of respiratory epithelium. Approach: Human primary respiratory epithelial cells are cultured in microfluidic chambers combined with a custom-made bioreactor (setup already established in our lab). This facilitates cell growth at the air-liquid interface and simultaneous mechanical stimulation with a linear actuator-driven membrane pump. Various patterns, stimulation periods, as well as long-term physiological and non-physiological flow patterns, are simulated by the air or liquid stream. To analyze the effect of different flow patterns, direction and coordination of cilia movement are evaluated by particle tracking of fluorescent microbeads. Epithelial differentiation, junctional communication and inflammatory responses are assessed by immunostainings, electron microscopy and ELISAs. To apply the results to tissue engineering of the airways, a 3D tubular construct is developed and dynamically stimulated in vitro to induce directed ciliary beating. We will also explore the possibilities of 3D model production technologies (ZWBF).